Effect of vitamin D pretreatment of human mesenchymal stem cells on ectopic bone formation.

Adult mesenchymal stem cells (MSCs) are used in contemporary strategies for tissue engineering. The MSC is able to form bone following implantation as undifferentiated cells adherent to hydroxyapatite (HA)/tricalcium phosphate (TCP) scaffolds. Previous investigators have demonstrated that human MSCs (hMSCs) can be differentiated to osteoblasts in vitro by the inclusion of vitamin D and ascorbic acid. The aim of this study was to compare the osteogenic potential of predifferentiated and undifferentiated bone marrow-derived, culture-expanded hMSCs adherent to synthetic HA/TCP (60%/40%) following subcutaneous engraftment in severe combined immunodeficiency (SCID) mice. During the final 3 days of culture, cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and antibiotics or media containing 25-mM calcium supplementation with vitamin D and ascorbic acid. Four weeks following implantation in SCID mice, scoring analysis of bone formation within the cubes revealed the absence of bone formation in unloaded cubes. Bone formation compared by a qualitative bone index was 7.23% for undifferentiated cells compared to 5.20% for differentiated cells. Minimal resorption was observed at this early time point. In this ectopic model, predifferentiation using a combination of vitamin D and ascorbic acid failed to increase subsequent bone formation by implanted cells. Following implantation of hMSCs adherent to an osteoconductive scaffold, host factors may contribute dominant osteoinductive signals or impose inhibitory signals to control the fate of the implanted cell. Predifferentiation strategies require confirmation in vivo.

In vitro characterization of mesenchymal stem cell-seeded collagen scaffolds for tendon repair: effects of initial seeding density on contraction kinetics.

Mesenchymal stem cells (MSCs) were isolated from bone marrow, culture-expanded, and then seeded at 1, 4, and 8 million cells/mL onto collagen gel constructs designed to augment tendon repair in vivo. To investigate the effects of seeding density on the contraction kinetics and cellular morphology, the contraction of the cell/collagen constructs was monitored over time up to 72 h in culture conditions. Constructs seeded at 4 and 8 million cells/mL showed no significant differences in their gross appearance and dimensions throughout the contraction process. By contrast, constructs seeded at 1 million cells/mL initially contracted more slowly and their diameters at 72 h were 62 to 73% larger than those seeded at higher densities. During contraction, MSCs reoriented and elongated significantly with time. Implants prepared at higher seeding densities showed more well aligned and elongated cell nuclei after 72 h of contraction. Changes in nuclear morphology of the MSCs in response to physical constraints provided by the contracted collagen fibrils may trigger differentiation pathways toward the fibroblastic lineage and influence the cell synthetic activity. Controlling the contraction and organization of the cells and matrix will be critical for successfully creating tissue engineered grafts.

Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair.

This investigation tested the hypothesis that delivering mesenchymal stem cell-seeded implants to a tendon gap model results in significantly improved repair biomechanics. Cultured, autologous, marrow-derived mesenchymal stem cells were suspended in a collagen gel delivery vehicle; the cell-gel composite was subsequently contracted onto a pretensioned suture. The resulting tissue prosthesis was then implanted into a 1-cm-long gap defect in the rabbit Achilles tendon. Identical procedures were performed on the contralateral tendon, but only the suture material was implanted. The tendon-implant constructs were evaluated 4, 8, and 12 weeks later by biomechanical and histological criteria. Significantly greater load-related structural and material properties were seen at all time intervals in the mesenchymal stem cell-treated tendons than in the contralateral, treated control repairs (p < 0.05), which contained suture alone with natural cell recruitment. The values were typically twice those for the control tissues at each time interval. Load-related material properties for the treated tissues also increased significantly over time (p < 0.05). The treated tissues had a significantly larger cross-sectional area (p < 0.05), and their collagen fibers appeared to be better aligned than those in the matched controls. The results indicate that delivering mesenchymal stem cell-contracted, organized collagen implants to large tendon defects can significantly improve the biomechanics, structure, and probably the function of the tendon after injury.

Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel.

Full-thickness articular cartilage defects are a major clinical problem; however, presently there is no treatment available to regeneratively repair these lesions. The current therapeutic approach is to drill the base of the defect to expose the subchondral bone with its cells and growth factors. This usually results in a repair tissue of fibrocartilage that functions poorly in the loaded joint environment. The use of phenotypically appropriate chondrocytes embedded in a collagen gel delivery vehicle may provide a method that could be used to repair full-thickness articular cartilage defects with functionally satisfactory hyaline cartilage. Allograft articular chondrocytes embedded in a type I collagen gel were transplanted into large (6 x 3 x 3 mm), full-thickness articular cartilage defects in condylar and patellar weight-bearing surfaces to develop clinically applicable methods to repair articular cartilage defects. Chondrocytes were isolated from the articular cartilage of 4-week-old New Zealand rabbits and embedded in type I collagen gels. This composite was transplanted into a full-thickness defect on the medial femoral condyle and patellar groove of adolescent host rabbits. The repair cartilage was assessed histologically by a semiquantitative scoring system and biomechanically with a microindentation technique of specimens 4-48 weeks after chondrocyte transplantation. Defects in both locations were repaired with histologically apparent hyaline cartilage observed from as early as 4 weeks until 48 weeks after transplantation. The repair cartilage in the medial femoral condyle was more irregular than in the patellar groove, but in all other respects was similar. The grafted tissue did not remodel and differentiate into the morphological zones seen in normal articular cartilage. No tidemark or subchondral bony plate formed even 48 weeks after transplantation. Biomechanically, the repaired cartilage demonstrated indentation values similar to normal articular cartilage 12 weeks after transplantation and remained the same 48 weeks after transplantation. By contrast, the control (i.e., empty) defects healed with tissue that exhibited very poor metachromatic staining and exhibited very high indentation values. Incomplete bonding of the repair tissue to the normal cartilage was seen, and the surface was significantly irregular with major discontinuities. These observations provide the basis for considering the use of allograft articular chondrocytes to repair articular cartilage defects in the weight-bearing regions of the knee.

Catastrophic antiphospholipid syndrome: response to repeated plasmapheresis over three years.

The catastrophic antiphospholipid syndrome (CAPS) is rare and usually fatal. In this report, we describe an unusual patient who, 31 years after experiencing an atypical preeclampsia-eclampsia presentation known today as the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), developed CAPS, which seemed to complicate a diagnosis of primary antiphospholipid syndrome. She responded to repeated plasmapheresis over 3 years. Anticoagulants, corticosteroids, intravenous gamma globulin, and intravenous cyclophosphamide had all failed to halt the progression of CAPS, but repeated plasmapheresis not only halted the condition, but it led to the reversal of a leukoencephalopathy. The relationship between HELLP syndrome and CAPS is discussed, and possible pathogenetic mechanisms that explain the efficacy of repeated plasmapheresis in this setting are suggested. It is postulated that perhaps plasmapheresis, through removal of cytokines or other mediators, disrupts the interaction between phospholipid-protein complexes and endothelial cells. Repeated plasmapheresis should be considered in the most refractory cases of CAPS when more conventional treatment regimens have failed.

Culture expanded canine mesenchymal stem cells possess osteochondrogenic potential in vivo and in vitro.

Mesenchymal Stem Cells (MSCs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes, myoblasts, and adipocytes have been previously isolated from the marrow and periosteum of human, murine, lapine, and avian species. This study documents the existence of similar multipotential stem cells in canine marrow. The cells were isolated from marrow aspirates using a modification of techniques previously established for human MSCs (hMSCs), and found to possess similar growth and morphological characteristics, as well as osteochondrogenic potential in vivo and in vitro. On the basis of these results, the multipotential cells that were isolated and culture expanded are considered to be canine MSCs (cMSCs). The occurrence of cMSCs in the marrow was determined to be one per 2.5 x 10(4) nucleated cells. After enrichment of the cMSCs by centrifugation on a Percoll cushion, the cells were cultivated in selected lots of serum. Like the hMSCs, cMSCs grew as colonies in primary culture and on replating, grew as a monolayer culture with very uniform spindle morphology. The population doubling time for these cMSCs was approximately 2 days. The morphology and the growth kinetics of the cMSCs were retained following repeated passaging. The osteogenic phenotype could be induced in the cMSC cultures by the addition of a synthetic glucocorticoid, dexamethasone. In these osteogenic cultures, alkaline phosphatase activity was elevated up to 10-fold, and mineralized matrix production was evident. When cMSCs were loaded onto porous ceramics and implanted in autologous canine or athymic murine hosts, copious amounts of bone and cartilage were formed in the pores of the implants. The MSC-mediated osteogenesis obtained by the implantation of the various MSC-loaded matrix combinations is the first evidence of osteogenesis in a canine model by implantation of culture expanded autologous stem cells. The identification and isolation of cMSCs now makes it feasible to pursue preclinical models of bone and cartilage regeneration in canine hosts.

A chemically defined medium supports in vitro proliferation and maintains the osteochondral potential of rat marrow-derived mesenchymal stem cells.

Among the stromal elements in mammalian and avian bone marrow there exists a pluripotent subset of cells which we refer to as mesenchymal stem cells (MSCs). These cells can be isolated and will proliferate in culture. When such subcultured cells are introduced into porous tricalcium phosphate-hydroxyapatite ceramic cubes and implanted subcutaneously into syngeneic or immunocompromised hosts, the passaged MSCs are observed to differentiate into bone and cartilage. Heretofore, those assays have been conducted with MSCs which had been maintained in vitro in serum-containing medium. A serum-free medium (RDM-F), which consists of insulin, 5 micrograms/ml, linoleic acid-bovine serum albumin, 0.1%, platelet-derived growth factor-BB, 10 ng/ml, and basic fibroblast growth factor, 1 ng/ml in a base medium of 60% Dulbecco's modified Eagle's medium with low glucose and 40% MCDB-201, has been developed for rat marrow-derived MSCs. Proliferation rates of MSCs maintained in RDM-F equal those of cells maintained in serum-containing medium through Day 4 following subculturing and continue at up to 80% of the rate of the latter through Day 8 of subculture. When tested in the in vivo ceramic cube assay, MSCs cultured in RDM-F retain their osteochondral potential and differentiate into bone and cartilage in a manner indistinguishable from those cultivated in serum-containing medium. Utilization of this serum-free medium will facilitate analysis of the effects of other growth factors and cytokines on the proliferation and differentiation of MSCs, without the complexity of exogenous serum.

Myogenic Expression of Mesenchymal Stem Cells within Myotubes of mdx Mice in Vitro and in Vivo.

The myogenic potential of bone marrow- and periosteum-derived mesenchymal stem cells (MSCs) was studied in vitro by coculture of MSCs of snj mice with myoblasts of newborn snj mice or 3-week-old mdx mice. MSCs were labeled with [(3)H]thymidine and cocultured with muscle precursor cells. At 5 different time points, the cocultures were harvested and prepared for autoradiography. Cocultures of MSCs and mdx mouse-derived myoblasts were immunostained for dystrophin before autoradiography. Autoradiographic grains were detected over isolated nuclei in myotubes, which stained positively with antidystrophin antibody. In vivo myogenic potential of MSCs was tested by direct injection into growing muscle of mdx mice. Equal numbers of nonmutant bone marrow-derived MSCs or myoblasts were injected separately into the tibialis anterior muscles of mdx mice. Muscle samples were harvested at 6, 8, and 10 weeks after injection, weighed, and stained with antidystrophin antibody. A small yet significant increase in muscle mass was observed in both the myoblast-injected (11% increase) and MSC-injected muscles (3%), as compared to controls. Muscle injected with myoblasts showed a remarkable conversion from dystrophin-negative to dystrophin-positive fibers (30-40%) in mdx mice injected with normal myoblasts, as previously reported by others. The frequency of dystrophin-positive fibers in mdx mouse muscle injected with marrow-derived MSCs was lower than that of the muscles injected with myoblasts, but was significantly higher than control muscles injected with medium. These results suggest that within the population of MSCs there are cells that are able to differentiate into skeletal muscle.

Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage.

UNLABELLED: Osteochondral progenitor cells were used to repair large, full-thickness defects of the articular cartilage that had been created in the knees of rabbits. Adherent cells from bone marrow, or cells from the periosteum that had been liberated from connective tissue by collagenase digestion, were grown in culture, dispersed in a type-I collagen gel, and transplanted into a large (three-by-six-millimeter), full-thickness (three-millimeter) defect in the weight-bearing surface of the medial femoral condyle. The contralateral knee served as a control: either the defect in that knee was left empty or a cell-free collagen gel was implanted. The periosteal and the bone-marrow-derived cells showed similar patterns of differentiation into articular cartilage and subchondral bone. Specimens of reparative tissue were analyzed with use of a semiquantitative histological grading system and by mechanical testing with employment of a porous indenter to measure the compliance of the tissue at intervals until twenty-four weeks after the operation. There was no apparent difference between the results obtained with the cells from the bone marrow and those from the periosteum. As early as two weeks after transplantation, the autologous osteochondral progenitor cells had uniformly differentiated into chondrocytes throughout the defects. This repair cartilage was subsequently replaced with bone in a proximal-to-distal direction, until, at twenty-four weeks after transplantation, the subchondral bone was completely repaired, without loss of overlying articular cartilage. The mechanical testing data were a useful index of the quality of the long-term repair. Twenty-four weeks after transplantation, the reparative tissue of both the bone-marrow and the periosteal cells was stiffer and less compliant than the tissue derived from the empty defects but less stiff and more compliant than normal cartilage. CLINICAL RELEVANCE: The current modalities for the repair of defects of the articular cartilage have many disadvantages. The transplantation of progenitor cells that will form cartilage and bone offers a possible alternative to these methods. As demonstrated in this report, autologous, bone-marrow-derived, osteochondral progenitor cells can be isolated and grown in vitro without the loss of their capacity to differentiate into cartilage or bone. Sufficient autologous cells can be generated to initiate the repair of articular cartilage and the reformation of subchondral bone. The repair tissues appear to undergo the same developmental transitions that originally led to the formation of articular tissue in the embryo.(ABSTRACT TRUNCATED AT 400 WORDS)

Osteogenesis in marrow-derived mesenchymal cell porous ceramic composites transplanted subcutaneously: effect of fibronectin and laminin on cell retention and rate of osteogenic expression.

Cultured-expanded rat marrow-derived mesenchymal cells differentiate into osteoblasts when combined with a porous calcium phosphate delivery vehicle and subsequently implanted in vivo. In this study, the effects of ceramic pretreatment with the cell-binding proteins fibronectin and laminin on the osteogenic expression of marrow-derived mesenchymal cells were assessed by scanning electron microscopy, [3H]-thymidine-labeled cell quantitation, and histological evaluation of bone formation. Scanning electron microscopic observations showed that marrow-derived mesenchymal cells rapidly spread and attach to both fibronectin- or laminin-adsorbed ceramic surfaces but retain a rounded morphology on untreated ceramic surfaces. Quantitation of [3H]-thymidine labeled cells demonstrated that laminin and fibronectin preadsorbed ceramics retain approximately double the number of marrow-derived mesenchymal cells than do untreated ceramics harvested 1 wk postimplantation. Histological observations indicate that the amount of time required to first detect osteogenesis was shortened significantly by pretreatment of the ceramic with either fibronectin or laminin. Fibronectin- and laminin-coated ceramic composite samples were observed to contain bone within 2 wk postimplantation, while in untreated ceramic the earliest observation of bone was at 4 wk postimplantation. A comparison was made of the initial cell-loading, in vivo cell retention characteristics, and rate of osteogenesis initiation of marrow-derived mesenchymal cells on two types of ceramic with different pore structure and chemical composition, with and without preadsorption with fibronectin or laminin. "Biphasic" ceramics contain randomly distributed pores 200-400 microns in diameter, and "coral-based" ceramics have continuous pores of approximately 200 microns in diameter. Laminin or fibronectin preadsorption significantly increases the number of cells retained in all ceramic test groups by day 7 postimplantation. In addition, by day 7 postimplantation, the biphasic ceramics retain a significantly greater number of cells for all test groups than do coral-based ceramics. The biphasic ceramics consistently have more specimens positive for bone with the identical cell-loading conditions used throughout this study. These results indicate that the retention of cells within the ceramic is an important factor for optimization of marrow mesenchymal cell initiated bone formation. The retention of cells within ceramics is augmented by the adsorption of the cell-binding proteins laminin and fibronectin, but this effect varies depending on ceramic pore structure and/or chemical composition.

The effects of Rumalon, a glycosaminoglycan peptide complex, in a partial meniscectomy model of osteoarthritis in rabbits.

The effects of Rumalon, a glycosaminoglycan peptide complex (GP-C), were evaluated in a partial meniscectomy model of osteoarthritis (OA). When rabbits were treated with Rumalon, 0.25 ml/kg 3 times/week for 12 weeks after partial meniscectomy, the proportion of animals that developed cartilage ulceration was lower than that in untreated meniscectomized rabbits (p less than 0.05). At 6 weeks sacrifice, the same dose of Rumalon was associated with a trend toward reduction in the number of animals with ulcerations. Rumalon administered at a dose of 0.5 ml/kg for 12 weeks reduced the number of animals with ulcers, although not at the p less than 0.05 level of significance. Semiquantitative histologic evaluations revealed no differences between control and treatment groups either in the thickness of articular cartilage, density of cells present, safranin-O stain or ratio of DNA/protein. Increased proteoglycan synthesis characteristic of OA was modulated toward normal in association with Rumalon therapy. Diminished OA changes in response to Rumalon are consistent with an ameliorative response to this agent in the partial meniscectomy model in the rabbit.

Early CT findings of global central nervous system hypoperfusion.

The early computed tomographic (CT) findings of acute global central nervous system hypoperfusion were studied in 10 patients. The findings could be characterized as: (1) diffuse mass effect with effacement of the cerebral sulci and of the brainstem cisterns (nine patients); (2) global decrease in the cortical gray-matter density from edema, causing loss of the normal gray-white matter differentiation (six patients); (3) low-density lesions of the basal ganglia bilaterally (five patients); and (4) decreased gray-matter density in watershed distributions bilaterally (two patients). Subsequent contrast-enhanced scans in three of the 10 patients demonstrated selective enhancement of the cerebral cortex or the basal ganglia or both. The CT findings seen in this study predicted a poor outcome; nine of the 10 patients died from the insult. The abnormal CT findings can be ascribed to increased vulnerability of the cerebral cortex and basal ganglia to hypotensive episodes. This vulnerability is due to the large metabolic demand of these regions and their characteristic local cerebral blood flow.

Computer analysis of processor status.

The authors describe the use of a desk-top computer to analyze processor status. Quality control data (sensitometry, densitometry, thermometry, etc.) are collected conventionally. Analysis of the data is subsequently performed using specially designed software. This computer analysis results in a small savings in time and in greatly improved management and presentation of quality control data.

Age and oxygen toxicity related fluorescence in the honey bee thorax.

The blue fluorescent material in thoraxes of honey bees of known adult age from eclosion to 28 days was measured. The amount was greater in older insects and exposure to ozone (ppm) augmented the rate of accumulation. The nearly constant daylight flying by the bees and the ozone effect suggest radical peroxidative origin of the fluorescence. The material was partially purified by chromatography. It is a relatively non-polar lipid.

Fluorescent age pigment in insect lysosomes.

Fluorescent pigment accumulates in the lysosomal fraction of spodoptera sixth instars. It was purified by silicic acid chromatography. The fluorescent fraction was characterized by conjugated unsaturation, reactivity with thiobarbituric acid, and lability in alkali; all reminiscent of age pigment. The phosphorus content of the purified fraction was negligible and nitrogen was not detected. It is suggested that the material is degraded, perhaps polymerized lipid fragments.

Economic impact of quality control in diagnostic radiology.

With ever-increasing costs confronting health care facilities, much attention is being focused on various cost-containment projects. Although it is generally assumed that a radiology department quality control program will increase costs, institutional savings may also result. A comprehensive quality control program involving continuing education, simplification of technic, processor control, standardization of cassettes, and preventive maintenance resulted in substantial savings for a medical school radiology department.

X-ray attenuation by prescription lenses.

The degree of attenuation of diagnostic x-rays by prescription lenses is reported. Photo-gray lenses provide considerable eye protection.

Interactions of standard antibody with Australia antigens in Au Ag-Ab radioimmunoassay.

Human antisera against Australia (Au) antigen have been characterized by liquid-phase radioimmunoassay (RIA) for their precipitation of (125)I-labeled Au antigen. The end-point dilutions of sera (anti-Au) which precipitated 50% of (125)I-Au antigen by RIA correlated well with complement fixation titers but had a much wider range, indicating a greater precision and perhaps a better sensitivity of assay. Anti-Au serum diluted to precipitate 50% of (125)I-labeled Au antigen was used as standard antibody in RIA tests to detect either inhibition or enhancement of the reaction by preincubated mixtures of Au antigen and antibody specimens. Without free Au antigen or antibody in the resultant mixtures there was no inhibition or enhancement; the mixtures presumably contained immunoreactively equivalent proportions of Au antigen and antibody. RIA data for diagnostic specimens indicated an end-point sensitivity which was proportional to the dilution of the standard anti-Au sera used in the test. High concentrations of the standard antibody permitted detectable inhibition of (125)I-Au antigen precipitation at lower antigen specimen concentrations. Similarly, low concentrations of the standard antibody permitted detectable enhancement of (125)I-Au antigen precipitation at lower antibody specimen concentrations. Omitting the standard antibody altogether resulted in a more sensitive RIA for Au antibody in test sera.