Mesenchymal stem cells: therapeutic outlook for stroke

Adult bone marrow-derived mesenchymal stem cells (MSCs) display a spectrum of functional properties. Transplantation of these cells improves clinical outcome in models of cerebral ischemia and spinal cord injury via mechanisms that may include replacement of damaged cells, neuroprotective effects, induction of axonal sprouting, and neovascularization. Therapeutic effects have been reported in animal models of stroke after intravenous delivery of MSCs, including those derived from adult human bone marrow. Initial clinical studies on intravenously delivered MSCs have now been completed in human subjects with stroke. Here, we review the reparative and protective properties of transplanted MSCs in stroke models, describe initial human studies on intravenous MSC delivery in stroke, and provide a perspective on prospects for future progress with MSCs.     

Treatment of osteoporosis with human parathyroid peptide and observations on effect of sodium fluoride.

OBJECTIVE--To evaluate the need for a randomised study of treatment of spinal osteoporosis with human parathyroid peptide in the secondary prevention of crush fractures; to study the effect of human parathyroid hormone peptide 1-34 plus sex hormones on vertebral body cancellous bone; and, separately, to determine the effect of relatively low doses of sodium fluoride plus calcium on spinal bone mineral density. DESIGN--Open study of patients with primary or postmenopausal osteoporosis. All patients had serial bone densitometry of the spine by quantitative computed tomography and dual photon absorptiometry as well as serial densitometry of the radial midshaft (cortical) and radial distal (trabecular) bone by quantitative computed tomography. Changes in the spinal bone not forming the spongiosa of the vertebral bodies ("cortical" bone) were determined from the difference between the two axial measurements, after correction to the same units of measurement. SETTING--Northwick Park Hospital and Medical Research Council Clinical Research Centre. PATIENTS--24 Patients who fulfilled the conventional criteria for type 1 (vertebral) osteoporosis not secondary to recognised causes other than sex hormone deficiency and with at least one crush or wedge vertebral fracture and a spinal bone density (quantitative computed tomography) less than 80 mg/cm3 or two or more fractures. Twelve patients received human parathyroid peptide and 12 sodium fluoride; they were not randomised. MAIN OUTCOME MEASURES--Trends in axial and peripheral bone mass values determined by linear, time dependent regression analyses. RESULTS--The patients receiving the peptide showed a substantial increase in vertebral spongiosa (mean 25.6 mg/cm2 two years after the start of treatment). No significant changes were seen in spinal cortical or radial bone density. The patients receiving sodium fluoride showed roughly equal increases in cancellous and cortical bone over the same period (mean increase in vertebral spongiosa 16.1 mg/cm3). No significant changes were seen in radial bone. CONCLUSIONS--Treatment of postmenopausal women with human parathyroid peptide selectively increases spinal cancellous bone density by amounts that may prove useful in secondary prevention. Peptide treatment should now be tested in a randomised study in which the important end point is prevention of fractures as the usefulness of sodium fluoride in this context is doubtful.     

Biomolecular strategies of bone augmentation in spinal surgery

Autologous bone grafts and allografts are the most accepted procedures for achieving spinal fusion. Recently, breakthroughs in understanding bone biology have led to the development of novel approaches to address the clinical problem of bone regeneration in an unfavorable environment, while bypassing the drawbacks of traditional treatments, including limited availability, donor site morbidity, risk of disease transmission and reduced osteogenicity. These approaches have also been studied for their effectiveness in reaching successful spinal fusion. This review focuses on the cellular and molecular mechanisms explaining the rationale behind these methods, including bone marrow aspirate and mesenchymal stem cells, platelet-rich plasma, bone morphogenetic proteins and gene therapy, which have opened a promising perspective in the field of bone formation in spinal surgery.     

Myelosuppressive Conditioning Using Busulfan Enables Bone Marrow Cell Accumulation in the Spinal Cord of a Mouse Model of Amyotrophic Lateral Sclerosis

Myeloablative preconditioning using irradiation is the most commonly used technique to generate rodents having chimeric bone marrow, employed for the study of bone marrow-derived cell accumulation in the healthy and diseased central nervous system. However, irradiation has been shown to alter the blood-brain barrier, potentially creating confounding artefacts. To better study the potential of bone marrow-derived cells to function as treatment vehicles for neurodegenerative diseases alternative preconditioning regimens must be developed. We treated transgenic mice that over-express human mutant superoxide dismutase 1, a model of amyotrophic lateral sclerosis, with busulfan to determine whether this commonly used chemotherapeutic leads to stable chimerism and promotes the entry of bone marrow-derived cells into spinal cord. Intraperitoneal treatment with busulfan at 60 mg/kg or 80 mg/kg followed by intravenous injection of green fluorescent protein-expressing bone marrow resulted in sustained levels of chimerism (∼80%). Bone marrow-derived cells accumulated in the lumbar spinal cord of diseased mice at advanced stages of pathology at both doses, with limited numbers of bone marrow derived cells observed in the spinal cords of similarly treated, age-matched controls; the majority of bone marrow-derived cells in spinal cord immunolabelled for macrophage antigens. Comparatively, significantly greater numbers of bone marrow-derived cells were observed in lumbar spinal cord following irradiative myeloablation. These results demonstrate bone marrow-derived cell accumulation in diseased spinal cord is possible without irradiative preconditioning.     

Is the vertebral canal prepared to host the aged spinal cord? A morphometric assessment

Although the interaction between the growing spinal cord and the vertebrae has been widely demonstrated for mammal’s prenatal and early postnatal life, there is no extensive knowledge about this interaction during late postnatal stages. It has been shown that spinal cord injuries are causally related to significant degenerative changes in bone properties. Nevertheless, information about a possible influence of the spinal cord on bone remodelling in adult healthy animals is missing. The aim of this research work was to assess possible morphological changes of the cervical vertebral canal of juvenile and aged rats during the ontogenetic period of adulthood that would justify the suggested influence. Since the spinal cord of rats increases its size with ageing, we analysed whether morphometric changes are occurring in the vertebral canal that would indicate bone remodelling in response to said growth. To this end, we used three complementary morphometric methods to describe the canal of the cervical and the first thoracic vertebrae. Geometric morphometric analyses evidence scarce variation in size and shape between juvenile and aged rats suggesting that, in general terms, the canal morphology of cervical vertebrae is already prepared in early adulthood to host the growing spinal cord. C3 was the only vertebra that showed consistent variation for the variables of canal thickness, perimeter, height and area. This regional variation may be linked to the patterns described for the changing spinal cord.     

Noggin is required for correct guidance of dorsal root ganglion axons

Members of the bone morphogenetic protein family of secreted protein signals have been implicated as axon guidance cues for specific neurons in Caenorhabditis elegans and in mammals. We have examined axonal pathfinding in mice lacking the secreted bone morphogenetic protein antagonist Noggin. We have found defects in projection of several groups of neurons, including the initial ascending projections from the dorsal root ganglia, motor axons innervating the distal forelimb, and cranial nerve VII. The case of the dorsal root ganglion defect is especially interesting: initial projections from the dorsal root ganglion enter the dorsal root entry zone, as normal, but then project directly into the gray matter of the spinal cord, rather than turning rostrally and caudally. Explant experiments suggest that the defect lies within the spinal cord and not the dorsal root ganglion itself. However, exogenous bone morphogenetic proteins are unable to attract or repel these axons, and the spinal cord shows only very subtle alterations in dorsal-ventral pattern in Noggin mutants. We suggest that the defect in projection into the spinal cord is likely the result of bone morphogenetic proteins disrupting the transduction of some unidentified repulsive signal from the spinal cord gray matter.     

Optimal condition of heparin-conjugated fibrin with bone morphogenetic protein-2 for spinal fusion in a rabbit model

Background aimsHeparin-conjugated fibrin (HCF) is a carrier for long-term release of bone morphogenetic protein-2 (BMP-2) and has been shown to promote bone formation in animal models. We performed an experimental study to determine the optimal dose of BMP-2 with an HCF carrier that promotes bone formation comparable to that of autograft while minimizing complications in spinal fusion.MethodsTwenty-four rabbits underwent posterolateral fusion of the L5–6 spinal segments. Different concentrations of HCF BMP-2 (1/10, 1/20, 1/30 or 1/40) were implanted in the spines of experimental rabbits, and autograft or INFUSE was implanted in the spines of control animals. Eight weeks after treatment, spinal fusion efficacy was evaluated by plain radiography, micro-computed tomography (micro-CT), mechanical testing and histomorphometry.ResultsSimilar to autograft, the 1/40 HCF BMP-2 showed significant bone formation on micro-CT and histomorphometry with mechanical stability. However, the other HCF BMP-2 concentrations did not show significant bone formation compared with autograft. Although conventional BMP-2 (INFUSE) led to higher bone formation and stability, it also led to excessive ectopic bone and fibrous tissue formation.ConclusionsThis study suggests the optimal concentration of BMP-2 using HCF for spinal fusion, which may decrease the complications of high-dose conventional BMP-2.     

A model for the autosensitization autoantibody production associated with xenogeneic thymic RNA.

Normal C57BL/6 bone marrow cells cultured for 3 weeks with xenogeneic thymic RNA and syngeneic C57BL/6 antigens (immunoglobulin G or red blood cells) produced anti-immunoglobulin antibody or anti-mouse red blood cell antibody (hemolysin). Addition of both xenogeneic thymic RNA and autoantigens to bone marrow cultures was necessary to elicit autosensitization. Syngeneic thymic RNA would not substitute for xenogeneic RNA. Normal recipients inoculated with syngeneic kidney or spinal cord homogenates and xenogeneic thymic RNA developed albuminuria or motor neuropathies within 10 days. Histologic examination of tissues from these animals revealed immunoglobulin deposits on glomerular or tubular basement membranes or on myelin sheaths. These changes were not observed in tissues from control animals inoculated with only the organ homogenates. Normal mice injected with syngeneic bone marrow cells, which had been autosensitized in vitro against kidney or spinal cord homogenates, also developed albuminuria or motor neuropathies, respectively. These abnormalities were observed only if bone marrow cells had been cultured with both xenogeneic thymic RNA and autoantigens. Histologic examination of tissues from these mice also revealed immunoglobulin deposits in kidney or spinal cord tissues. These results demonstrate that xenogeneic thymic RNA can play important roles in the formation of autoantibodies.     

From tendon to nerve: an MSC for all seasons

The potential of mesenchymal stem cells (MSCs) to regenerate damaged tissue is well documented, as this specialized progenitor cell type exhibits superior cellular properties, and would allow medical as well as ethical limitations to be overcome. By now, MSCs have been successfully introduced in manifold experimental approaches within the newly defined realm of Regenerative Medicine. Advanced methods for in vitro cell expansion, defined induction of distinct differentiation processes, 3-dimensional culture on specific scaffold material, and tissue engineering approaches have been designed, and many clinical trials not only have been launched, but recently could be completed. To date, most of the MSC-based therapeutic approaches have been executed to address bone, cartilage, or heart regeneration; further, prominent studies have shown the efficacy of ex vivo expanded and infused MSCs to countervail graft-versus-host disease. Yet more fields of application emerge in which MSCs unfold beneficial effects, and presently, therapies that effectively ameliorate nonhealing conditions after tendon or spinal cord injury are, courtesy of scientific research, forging ahead to the clinical trial stage.     

The clinical relevance of calcaneus bone mineral measurements: a review

The use of bone mineral content (BMC) measurements to assist in the management of osteoporosis has received increasing emphasis in recent years. Although the calcaneus, an essentially trabecular bone (90–95%), has been used extensively in the NASA experiments, few data relating to primary osteoporosis have appeared in the literature until recently. This paper is a review of the methods of measurement, their precision and methods of calibration, and the relationship of calcaneal mineral content to age, height, weight, other bone sites, degree of spinal osteoporosis, metabolic bone disease and the effects of therapeutic drugs. Prospectively, calcaneal BMC relates as well as spinal BMC to osteoporotic fracture risk. It is this use of BMC measurements which has the most promise for the future. The data indicate that osteoporosis is a systemic disease and trabecular bone losses are reflected in the calcaneus as well as in the spine itself.     

Spinal cord injury-related bone impairment and fractures: an update on epidemiology and physiopathological mechanisms

A sudden loss of motor function in segments of the spinal cord results in immobilisation and is complicated by bone loss and fractures in areas below the level of injury. Despite the acceptance of osteoporosis and fractures as two major public health problems, in people with spinal cord injuries, the mechanisms are not adequately investigated. Multiple risk factors for bone loss and fractures are present in this disabled population. This review is an update on the epidemiology and physiopathological mechanisms in spinal cord injury-related bone impairment and fractures.     

Effect of strontium substituted ß-TCP associated to mesenchymal stem cells from bone marrow and adipose tissue on spinal fusion in healthy and ovariectomized rat

Despite alternatives to autogenous bone graft for spinal fusion have been investigated, it has been shown that osteoconductive materials alone do not give a rate of fusion comparable with autogenous bone. This study analyzed a strontium substituted ß-tricalcium phosphate (Sr-ßTCP) associated with syngeneic, unexpanded, and undifferentiated mesenchymal stem cells from bone marrow (BMSC) or adipose tissue (ADSC) as a new tissue engineering approach for spinal fusion procedures. A posterolateral fusion was performed in 15 ovariectomized (OVX) and 15 sham-operated (SHAM) Inbred rats. Both SHAM and OVX animals were divided into three groups: Sr-ßTCP, Sr-ßTCP + BMCSs, and Sr-ßTCP + ADSCs. Animals were euthanized 8 weeks after surgery and the spines evaluated by manual palpation, micro-CT, and histology. For both SHAM and OVX animals, the fusion tissue in the Sr-ßTCP + BMSCs group was more solid. This effect was significantly higher in OVX animals by comparing the Sr-ßTCP + BMCSs group with Sr-ßTCP + ADSCs. Radiographical score, based on micro-CT 2D image, highlighted that the Sr-ßTCP + BMCSs group presented a similar fusion to Sr-ßTCP and higher than Sr-ßTCP + ADSCs in both SHAM and OVX animals. Micro-CT 3D parameters did not show significant differences among groups. Histological score showed significantly higher fusion in Sr-ßTCP + BMSCs group than Sr-ßTCP and Sr-ßTCP + ADSCs, for both SHAM and OVX animals. In conclusion, our results suggest that addition of BMSCs to a Sr-ßTCP improve bone formation and fusion, both in osteoporotic and nonosteoporotic animal, whereas spinal fusion is not enhanced in rats treated with Sr-ßTCP + ADSCs. Thus, for conducting cells therapy in spinal surgery BMSCs still seems to be a better choice compared with ADSCs.     

Large Specimen Bone Embedment and Cement Line Staining

Undecalcified embedment of large bone specimens is often challenging. A method is presented here that is suitable for methacrylate embedment of sections of canine vertebrae while retaining the ability to localize tartrate-resistant acid phosphatase and alkaline phosphatase activity. Specimens also retained tetracycline labelling, and sectioned preparations were readily stained with routine bone procedures. A modification of the Bodian silver stain, used for examining the nerves and spinal cord in these specimens, provided a useful stain for canaliculi and cement lines in trabecular and cortical bone. This stain is advantageous when both bone and nerve tissue are of interest, as in spinal fusion studies.     

Linkage of a Gene Causing High Bone Mass to Human Chromosome 11 (11q12-13)

The purpose of this paper is to report the linkage of a genetic locus (designated "HBM") in the human genome to a phenotype of very high spinal bone density, using a single extended pedigree. We measured spinal bone-mineral density, spinal Z(BMD), and collected blood from 22 members of this kindred. DNA was genotyped on an Applied Biosystems model 377 (ABI PRISM Linkage Mapping Sets; Perkin Elmer Applied Biosystems), by use of fluorescence-based marker sets that included 345 markers. Both two-point and multipoint linkage analyses were performed, by use of affected/unaffected and quantitative-trait models. Spinal Z(BMD) for affected individuals (N = 12) of the kindred was 5.54 +/- 1.40; and for unaffected individuals (N = 16) it was 0.41 +/- 0.81. The trait was present in affected individuals 18-86 years of age, suggesting that HBM influences peak bone mass. The only region of linkage was to a series of markers on chromosome 11 (11q12-13). The highest LOD score (5.21) obtained in two-point analysis, when a quantitative-trait model was used, was at D11S987. Multipoint analysis using a quantitative-trait model confirmed the linkage, with a LOD score of 5.74 near marker D11S987. HBM demonstrates the utility of spinal Z(BMD) as a quantitative bone phenotype that can be used for linkage analysis. Osteoporosis pseudoglioma syndrome also has been mapped to this region of chromosome 11. Identification of the causal gene for both traits will be required for determination of whether a single gene with different alleles that determine a wide range of peak bone densities exists in this region.     

The Controversy Surrounding Bone Morphogenetic Proteins in the Spine: A Review of Current Research

Bone morphogenetic proteins have been in use in spinal surgery since 2002. These proteins are members of the TGF-beta superfamily and guide mesenchymal stem cells to differentiate into osteoblasts to form bone in targeted tissues. Since the first commercial BMP became available in 2002, a host of research has supported BMPs and they have been rapidly incorporated in spinal surgeries in the United States. However, recent controversy has arisen surrounding the ethical conduct of the research supporting the use of BMPs. Yale University Open Data Access (YODA) recently teamed up with Medtronic to offer a meta-analysis of the effectiveness of BMPs in spinal surgery. This review focuses on the history of BMPs and examines the YODA research to guide spine surgeons in their use of BMP in spinal surgery.     

Softening Hair with Dithiothreitol (Cleland's Reagent) for Histological Sectioning in Paraffin

Undecalcified embedment of large bone specimens is often challenging. A method is presented here that is suitable for methacrylate embedment of sections of canine vertebrae while retaining the ability to localize tartrate-resistant acid phosphatase and alkaline phosphatase activity. Specimens also retained tetracycline labelling, and sectioned preparations were readily stained with routine bone procedures. A modification of the Bodian silver stain, used for examining the nerves and spinal cord in these specimens, provided a useful stain for canaliculi and cement lines in trabecular and cortical bone. This stain is advantageous when both bone and nerve tissue are of interest, as in spinal fusion studies.     

Adult mesenchymal stem cells for tissue engineering versus regenerative medicine

Adult mesenchymal stem cells (MSCs) can be isolated from bone marrow or marrow aspirates and because they are culture-dish adherent, they can be expanded in culture while maintaining their multipotency. The MSCs have been used in preclinical models for tissue engineering of bone, cartilage, muscle, marrow stroma, tendon, fat, and other connective tissues. These tissue-engineered materials show considerable promise for use in rebuilding damaged or diseased mesenchymal tissues. Unanticipated is the realization that the MSCs secrete a large spectrum of bioactive molecules. These molecules are immunosuppressive, especially for T-cells and, thus, allogeneic MSCs can be considered for therapeutic use. In this context, the secreted bioactive molecules provide a regenerative microenvironment for a variety of injured adult tissues to limit the area of damage and to mount a self-regulated regenerative response. This regenerative microenvironment is referred to as trophic activity and, therefore, MSCs appear to be valuable mediators for tissue repair and regeneration. The natural titers of MSCs that are drawn to sites of tissue injury can be augmented by allogeneic MSCs delivered via the bloodstream. Indeed, human clinical trials are now under way to use allogeneic MSCs for treatment of myocardial infarcts, graft-versus-host disease, Crohn's Disease, cartilage and meniscus repair, stroke, and spinal cord injury. This review summarizes the biological basis for the in vivo functioning of MSCs through development and aging.     

Can periods of static loading be used to enhance the resistance of the spine to cumulative compression?

Results of in vitro studies conducted on isolated bone specimens have indicated a higher tolerance to static load than exists when exposed to cyclic loading, when controlled for creep rate. If this difference in load tolerance exists, it may be exploited to extend the life of vertebral bone exposed to repetitive compression, and potentially alter the development of spinal injury. However, little work has been conducted on functional spinal units to determine if bone displays this characteristic within an intact joint. Additionally, static loading may result in load redistribution within the intervertebral disc forcing more of the compressive load towards the periphery of the endplate away from the nucleus. In order to examine these potential mechanisms, 218 osteoligamentous porcine functional spinal units were assigned to one of 15 loading scenarios. This involved one of three normalized peak load magnitudes (50%, 70% and 90% of estimated compressive tolerance) and one of five normalized static load applications (0%, 50%, 100%, 200% and 1000% of the total dynamic work duration). Load magnitude significantly altered the resistance to cumulative compression with decreased peak magnitudes corresponding to both increased cumulative load tolerance and increased height loss. Static load periods did not alter the resistance of the spinal unit to cumulative compression or impact the number of cycles tolerated to failure. The insertion of static load periods impacted the total survival time to failure, but only for the 1000% static load group, an exposure unlikely to occur for most in vivo exposures. The insertion of static load periods decreased the amount of height loss during testing which may play a protective role by allowing load redistribution within the vertebral bone and intervertebral disc.     

Neuronal development: putting motor neurons in their place

In the developing spinal cord, motor neurons occupy discrete columns with different identities and axon projections. This organisation has now been shown to depend crucially on sequential phases of expression of Hox-c proteins, generated in response to fibroblast growth factor signals.     

An improved design of electrodes for measurement of streaming potentials on wet bone in vitro and in vivo

Streaming potentials are generated by mechanical stress in wet bone and may constitute a control mechanism for bone remodeling. Measurement of streaming potentials in bone has attracted considerable effort in past years but quantitative studies have been hampered by relatively poor repeatability when using Ag.AgCl electrodes which contact bone via a wick moistened with electrolyte. Improvement now has been achieved with an electrode design that limits the specific area of contact of an agar/salt bridge by means of a silastic seal, thus permitting the same equipotential surface to be contacted for each set of measurements. This reduces variations caused by bone structure and impedance, and facilitates quantitative comparisons of the response of bone samples to selected variables. The new design also permits considerable qualitative improvement in recordings made from bone during locomotor function in experimental animals in vivo.