Medial Unicompartmental Knee Arthroplasty for the Treatment of Focal Femoral Osteonecrosis.

BACKGROUND: Previous research has indicated that unicompartmental arthroplasty may be an effective treatment for focal osteonecrosis in the knee; however, these reports have been composed of small patient cohorts and without characterization of the osteonecrotic lesions. Therefore, the purpose of this study was to investigate the effectiveness of unicompartmental arthroplasty for the treatment of focal osteonecrosis within the medial femoral condyle including an assessment of lesion size. METHODS: A consecutive series of >5,000 unicompartmental knee arthroplasties performed at a single institution was retrospectively reviewed to identify cases of medial femoral condyle osteonecrosis with a minimum 2-year follow-up. Lesion size was classified according to the ratio of lesion width to condylar width, as well as lesion depth relative to condylar depth. Patient-reported outcome measures and need for a revision procedure were studied. RESULTS: Sixty-four patients (32 males, 32 females; 65 knees) with a mean age of 64 years were included. The mean patient follow-up was 5.3 years (range, 2 to 12 years). The mean ratio of lesion width to condylar width was 64%, the mean lesion depth was 1.11 cm, and 82% of cases demonstrated subchondral collapse. At the time of the latest follow-up, patients demonstrated substantial improvements in the pain, function, and clinical components of the Knee Society Score, by 36, 25, and 51, respectively. Four patients (6%) required a revision, of which only 1 was for aseptic loosening of the femoral component. CONCLUSIONS: Unicompartmental arthroplasty is an effective treatment for advanced-stage focal osteonecrosis of the medial femoral condyle. Loss of component fixation to the femoral condyle did not appear to be a substantial concern because there was only 1 femoral failure as a result of aseptic loosening, despite lesions affecting a significant portion of the femoral condyle. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Reduced Thigh Pain with Short Femoral Stem Design Following Direct Anterior Primary Total Hip Arthroplasty.

BACKGROUND: Thigh pain is a variably reported symptom in the postoperative period following primary total hip arthroplasty (THA) with a well-fixed cementless femoral implant. While research has identified stem size, stem alignment, and differences in modulus of elasticity between implant and host bone as potential sources of thigh pain, only one study has specifically examined the impact of variation in implant design within a single femoral stem design. The purpose of this work was to determine whether there were differences in the pain experienced by patients treated with two design variants of a femoral stem during direct anterior THA. MATERIALS AND METHODS: Patients undergoing primary direct anterior THA at a single center between 2011-2015 were included in the study. Those patients suffering extensive comorbidities and postoperative complications were excluded from analysis. Study subjects completed a pain drawing and scale questionnaire for the operative hip at least one year following surgery. A cementless, single-taper wedge, titanium femoral component design available in short- and standard-length variations was used in all cases. Pain outcomes were compared between these two femoral stem options. RESULTS: A total of 1347 patients (1536 THA) met inclusion criteria for the study and surveys were returned for 820 of these THAs. Demographic data and UCLA activity scores were similar between cohorts of patients receiving the short- and standard-length components. The most common locations of pain reported were in the lower back and trochanteric region, 28% and 24% respectively. Patients in the short-length cohort reported a significantly lower incidence of pain in the anterior thigh as compared to the standard-length cohort, 12% versus 19% respectively [p=0.007]. There was no difference in the number of patients experiencing moderate to severe intensity of anterior thigh pain between these two groups, 3% versus 5% respectively [p=0.36]. No other statistically significant differences were found in the incidence of pain in the lower back, buttock, groin, trochanter, lateral thigh, or posterior thigh regions between the two cohorts. CONCLUSION: While the lower back and trochanteric region may be the most frequent areas of pain experienced in patients at one-year or more postoperative from direct anterior THA, a significantly higher incidence of anterior thigh pain is found in those patients treated with a standard-length stem design as compared to the short design. This finding may be due to contact between the tip of the distal stem with the femoral diaphysis as has been theorized in previous research, which is circumvented with the short design variant.

Lateral Unicompartmental Knee Arthroplasty Utilizing a Modified Surgical Technique and Specifically Adapted Fixed-Bearing Implant.

BACKGROUND: Treatment of isolated lateral compartment arthritic disease with partial knee arthroplasty remains underutilized in comparison to medial unicompartmental arthroplasty. This study examines the survival and outcome of lateral unicompartmental arthroplasty utilizing the first implant specifically developed for the lateral compartment. MATERIALS AND METHODS: A retrospective review was performed to detect lateral unicompartmental arthroplasty procedures performed in our practice between January 2013 and May 2016. Patients indicated for surgery met specific preoperative clinical and radiographic criteria confirming lateral compartment arthritic disease with a correctable deformity, intact full-thickness medial cartilage, competent anterior cruciate ligament, and minimal disease in the patellofemoral compartment. A single implant design was used in all cases which consisted of a fixed-bearing tibial component specifically adapted to lateral compartment anatomy. Unicompartmental arthroplasty surgical technique was adjusted to attempt to recreate lateral compartment kinematics. RESULTS: Fifty-two consecutive patients (56 knees) with lateral unicompartmental arthroplasty procedures meeting minimum two-year follow up were included in the study. Thirty-nine patients were female, and 93% of cases were performed for treatment of osteoarthritis. At a mean follow up of nearly three years, Knee Society clinical and functional scores improved postoperatively by a mean difference of 41 and 21, respectively. There were two reoperations, one medial unicompartmental arthroplasty for osteoarthritis progression and a superficial debridement for a non-healing wound. Thus, failure of lateral unicondylar knee arthroplasty (UKA) was less than 2% in this study. There were no other component revisions, radiographic evidence of loosening, or clinical failures. CONCLUSIONS: At early follow up, lateral unicompartmental arthroplasty using a modified surgical technique and an implant specifically designed for the lateral compartment is a reliable treatment for isolated lateral femorotibial arthritis when meeting defined indications.

"Thicker" Polyethylene Bearings Are Not Associated With Higher Failure Rates in Primary Total Knee Arthroplasty.

BACKGROUND: Despite improvements in polyethylene bearing surface properties, only 1 previous study has examined the results of larger thickness bearings. The purpose of this study was to determine whether polyethylene thickness influenced patient outcomes and implant survival following modular total knee arthroplasty. METHODS: A retrospective review was performed of patients undergoing primary total knee arthroplasty from 2003 to 2014 in a single practice database. Patients were separated into "thin" and "thick" polyethylene groups based on manufacturer polyethylene bearing sizes of 14 mm or less compared to those greater than 14 mm, respectively. Patient clinical outcomes, need for revision surgery, and overall implant survival rates were evaluated. RESULTS: A total of 6698 primary knee arthroplasties were included, and a thin bearing was used in 96.5% of these cases. Preoperatively, patients with a thick bearing had significantly lower Knee Society clinical scores (P < .01), a trend toward lower functional scores (P = .06), and more significant coronal plane deformity. Postoperatively, patients with thick bearings exhibited better Knee Society clinical and pain scores as well as similar functional scores and University of California at Los Angeles activity scores. The overall reoperation rate and 10-year survivorship free of revision were similar between thick and thin bearings (1.7% vs 2.3%; 98.2% vs 96.1%). Patients with thin bearings were twice as likely to require a manipulation under anesthesia postoperatively (P = .02), while there were no failures in the thick bearing group due to aseptic loosening or instability. CONCLUSION: Patients with thick polyethylene bearings performed similarly or better in multiple clinical outcomes and survivorship compared to those with thin bearings.

Medial Mobile-Bearing Unicompartmental Knee Arthroplasty in Young Patients Aged Less Than or Equal to 50 Years.

BACKGROUND: Contemporary research has shown medial mobile-bearing unicompartmental knee arthroplasty to be an effective treatment in patients younger than 60 years; however, only one other study has specifically investigated unicompartmental arthroplasty outcomes in patients 50 years or younger. The purpose of this study was to determine the clinical outcomes and survivorship of medial mobile-bearing unicompartmental arthroplasty in this younger patient population. METHODS: A retrospective review of patients undergoing primary unicompartmental knee arthroplasty using the Oxford partial knee from 2003 to 2014 in a single practice database was performed. Patients were included in the study if they were 50 years of age or younger with a primary diagnosis of anteromedial osteoarthritis and minimum clinical follow-up of 2 years. Patient clinical outcomes, function, and need for revision surgery were assessed. RESULTS: The study included 340 knees. Average patient age was 46.5 years, and the mean follow-up was 6.1 years. Patients demonstrated significant improvements (P < .05) in range of motion (114.5 v 116.9), University of California Los Angeles activity score (4.4 vs 5.6), Knee Society clinical (37.3 vs 86.5) and functional scores (58.8 v 79.8). Overall, 20 patients required reoperation, and the predicted survival rate was 96% at 6 years and 86% at 10 years. Aseptic loosening occurred in 7 patients at an average of 5.6 years postoperatively, while 4 patients required conversion to total knee arthroplasty because of arthritic progression at a mean time of 6.6 years. There were no revision procedures required due to polyethylene liner wear or breakage. CONCLUSION: Medial mobile-bearing unicompartmental arthroplasty should be considered as a treatment option in patients younger than 50 years of age suffering from anteromedial osteoarthritis of the knee.

Posterior Shoulder Instability in Overhead Athletes.

Posterior shoulder instability in overhead athletes presents a unique and difficult challenge. Often, this group has an inherent capsular laxity and/or humeral retroversion to accommodate the range of motion necessary to throw. This adaptation makes the diagnosis of posterior capsulolabral pathology challenging, as the examiner must differentiate between adaptive capsular laxity and pathologic instability. Further complicating matters, the intraoperative surgeon must find the delicate balance of achieving stability while still allowing the necessary range of motion.

Paracrine proangiopoietic effects of human umbilical cord blood-derived purified CD133+ cells--implications for stem cell therapies in regenerative medicine.

CD133+ cells purified from hematopoietic tissues are enriched mostly for hematopoietic stem/progenitor cells, but also contain some endothelial progenitor cells and very small embryonic-like stem cells. CD133+ cells, which are akin to CD34+ cells, are a potential source of stem cells in regenerative medicine. However, the lack of convincing donor-derived chimerism in the damaged organs of patients treated with these cells suggests that the improvement in function involves mechanisms other than a direct contribution to the damaged tissues. We hypothesized that CD133+ cells secrete several paracrine factors that play a major role in the positive effects observed after treatment and tested supernatants derived from these cells for the presence of such factors. We observed that CD133+ cells and CD133+ cell-derived microvesicles (MVs) express mRNAs for several antiapoptotic and proangiopoietic factors, including kit ligand, insulin growth factor-1, vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8. These factors were also detected in a CD133+ cell-derived conditioned medium (CM). More important, the CD133+ cell-derived CM and MVs chemoattracted endothelial cells and display proangiopoietic activity both in vitro and in vivo assays. This observation should be taken into consideration when evaluating clinical outcomes from purified CD133+ cell therapies in regenerative medicine.

Umbilical cord blood-derived very small embryonic like stem cells (VSELs) as a source of pluripotent stem cells for regenerative medicine.

Umbilical cord blood-derived very small embryonic-like stem cells (UCB-VSELs) are the most primitive stem cells circulating in fetal peripheral blood. These very rare cells slightly smaller than red blood cells i) become mobilized during delivery, ii) are enriched in fraction of CD133+ Lin-CD45- cells iii) express markers of pluripotent stem cells (e.g., Oct4, Nanog, and SSEA-4) and iv) display a distinct morphology characterized by a high nuclear/ cytoplasmic ratio and undifferentiated chromatin. We envision that VSELs are released into neonatal peripheral blood as a migrating population of stem cells involved in regeneration of tissues that become damaged in the process of delivery. They may also be responsible for the occurrence of fetal-maternal chimerism. Our most recent data suggest that UCB-VSELs exhibit some characteristics of long-term repopulating hematopoietic stem cells (LT-HSCs). We propose that UCB-VSELs may eventually be employed as a source of pluripotent stem cells in regenerative medicine.

Safety and efficacy of bone marrow-derived autologous CD133+ stem cell therapy.

The Phase I clinical study was designed to assess the safety and feasibility of a dose escalating intracoronary infusion of autologous bone marrow (BM)-derived CD133+ stem cell therapy to the patients with chronic total occlusion (CTO) and ischemia. Nine patients were received CD133+ cells into epicardial vessels supplying collateral flow to areas of viable ischemic myocardium in the distribution of the CTO. There were no major adverse cardiac events (MACE), revascularization, re-admission to the hospital secondary to angina, or acute myocardial infarction (AMI) for the 24-month period following cellular infusion. In addition, there were no periprocedural infusion-related complications including malignant arrhythmias, loss of normal coronary blood flow or acute neurologic events. Cardiac enzymes were negative in all patients. There was an improvement in the degree of ischemic myocardium, which was accompanied by a trend towards reduction in anginal symptoms. Intracoronary infusion of autologous CD133+ marrow-derived cells is safe and feasible. Cellular therapy with CD133+ cells to reduce anginal symptoms and to improve ischemia in patients with CTO awaits clinical investigation in Phase II/III trials.

Responsiveness of the International Knee Documentation Committee Subjective Knee Form in comparison to the Western Ontario and McMaster Universities Osteoarthritis Index, modified Cincinnati Knee Rating System, and Short Form 36 in patients with focal articular cartilage defects.

BACKGROUND: The International Knee Documentation Committee Subjective Knee Form (IKDC SKF) is a patient-reported knee-specific outcome measure that has been shown to be a reliable, valid, and responsive measure for patients with a variety of knee conditions. Further testing is required to compare the reliability and responsiveness of the IKDC SKF to other commonly used patient-reported outcome measures for patients with articular cartilage lesions. HYPOTHESIS: The IKDC SKF has equal or better levels of reliability and responsiveness than the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), modified Cincinnati Knee Rating System (CKRS), and the Short Form 36 in patients with articular cartilage lesions. STUDY DESIGN: Cohort study (diagnosis); Level of evidence, 2. METHODS: Reliability was assessed by administering the 4 patient-reported outcome measures to 17 individuals who had undergone articular cartilage surgery 5 years before participation in this study. Responsiveness was determined by administering the 4 patient-reported outcome measures to 51 individuals with diagnosed focal articular cartilage defects who were scheduled to undergo surgical treatment. In both groups, the outcome measures were administered at baseline and at 6 and 12 months' follow-up. Participants also provided a global rating of change in comparison to baseline at the 6- and 12-month follow-ups. RESULTS: Test-retest reliability coefficients were 0.91 and 0.93 for the IKDC SKF at the 6- and 12-month follow-ups, respectively. The effect sizes and standardized response means were large (>0.80) at 6 months after surgery for the WOMAC pain, physical function, and total scores and 12 months after surgery for the IKDC SKF; WOMAC pain, physical function, and total; and CKRS scores. Six months after surgery, significant differences between those who were improved compared with those who were unchanged or worse were found only for the IKDC SKF. Twelve months after surgery, significant differences between the improved and unchanged groups were found for all of the knee-specific patient-reported outcome measures. Finally, the IKDC SKF, WOMAC, and CKRS scores were able to differentiate between individuals who perceived themselves to be improved versus not improved and the minimum clinically important difference for the IKDC SKF was 6.3 at 6 months and 16.7 at 12 months. CONCLUSION: The reliability and responsiveness of the IKDC SKF is comparable with other commonly used patient-reported outcome measures for patients with articular cartilage lesions. The IKDC SKF is a suitable alternative to other commonly used knee-specific instruments for measuring symptoms, daily function, and level of symptom-free sports activity in patients undergoing articular cartilage surgery.

Umbilical cord blood-selected CD133(+) cells exhibit vasculogenic functionality in vitro and in vivo.

BACKGROUND AIMS: Current clinical trials utilize non-selected bone marrow (BM) mononuclear cells (MNC) to augment vasculo genesis within ischemic vascular beds. Recent reports have identified a diminished number and function of hemat-opoietic stem cells (HSC) from aged and diseased patients. Umbilical cord blood (UCB) provides a potential robust allo-geneic source of HSC for therapeutic vasculogenesis. METHODS: MNC and magnetically isolated CD133(+) cells were assessed for viability (trypan blue) and surface phenotype (flow cytometry). To test in vivo functionality of the cells, NOD/SCID mice underwent ligation of the right femoral artery followed immediately by cell injection. Blood flow recovery, necrosis, BM engraftment of human cells and histologic capillary density were determined. Cells were tested for potential mechanisms mediating the in vivo effects, including migration, cytokine secretion and angiogenic augmentation (Matrigel assays). RESULTS: Surface expression analysis showed CD31 (PECAM) expression was greatly increased in UCB CD133(+) cells compared with BM MNC. At 28 days, perfusion ratios were highest in animals receiving UCB CD133(+) cells, while animals receiving BM CD133(+) cells and BM MNC demonstrated perfusion ratios statistically higher than in animals treated with cytokine media alone. Animals receiving CD133(+) cells showed a statistically higher capillary density, reduced severe digit necrosis and increased engraftment in the BM than animals treated with unselected BM MNC. In vitro studies showed equivalent migration to stromal-derived factor-1 (SDF-1), increased production of tumor necrosis factor alpha (TNF-alpha) and increased branch points with the co-incubation of CD133(+) cells with human umbilical vein endothelial cells (HUVEC) in the Matrigel angiogenesis assay. CONCLUSIONS: Taken together, UCB CD133(+) cells exhibit robust vasculogenic functionality compared with BM MNC in response to ischemia.

Furan Decorated Nucleoside Analogues as Fluorescent Probes: synthesis, photophysical evaluation and site-specific incorporation.

The synthesis and photophysical evaluation of modified nucleoside analogues in which a five-membered heterocycle (furan, thiophene, oxazole and thiazole) is attached to the 5 position of 2'-deoxyuridine are reported. The furan containing derivative is identified as the most promising responsive nucleoside of this family due to its emission quantum efficiency and degree of sensitivity to its microenvironment. The furan moiety was then attached to the 5 position of 2'-deoxycytidine as well as the 8 position of adenosine and guanosine. Photophysical evaluation of these four furan containing nucleoside analogues reveal distinct differences in the absorption, emission and quantum efficiency depending upon the class of nucleoside (pyrimidine or purine). Comparing the photophysical properties of all furan containing nucleosides, identifies the furan thymidine analogue, 5-(fur-2-yl)-2'-deoxyuridine, as the best candidate for use as a responsive fluorescent probe in nucleic acids. 5-(fur-2-yl)-2'-deoxyuridine was then converted to the corresponding phosphoramidite and site specifically incorporated into DNA oligonucleotides with greater than 88% coupling efficiency. Such furan-modified oligonucleotides form stable duplexes upon hybridization to their complementary DNA strands and display favorable fluorescent features.

The safety of autologous intracoronary stem cell injections in a porcine model of chronic myocardial ischemia.

BACKGROUND: Intracoronary mononuclear cell therapy may produce angiogenesis in chronic myocardial ischemia. Potential complications include periprocedural infarction secondary to: reduced coronary blood flow; hyperviscosity from the cell preparation; or microvascular dysfunction. To date, no studies to evaluate these potential complications have been reported. The objective of this report was to study the safety and feasibility of intracoronary injections of autologous bone marrow mononuclear cells in a porcine chronic myocardial ischemia model. METHODS: Domestic pigs (n = 5) underwent ameroid cuff placement of the left circumflex artery. Bone marrow-derived mononuclear cells [15 x 10(6) cells] labeled with CM dioctadecyl tetramethylindocarbocyanine were given by intracoronary injection. Animals were sacrificed, and hearts and vital organs were inspected grossly and by histopathology, and bone marrow underwent immunofluorescence microscopy. RESULTS: Troponin I levels, gross inspection and histopathology did not reveal evidence of myocardial infarction. Labeled cells were observed in perivascular structures in myocardium at the injection site in all animals and in the spleen from one animal. Bone marrow aspirates indicated labeled cells. CONCLUSIONS: Intracoronary injection of autologous mononuclear cells in a porcine chronic myocardial ischemia model appears safe. Intracoronary injection resulted in cell localization in the perivascular areas of myocardium supplied by the injected vessel. Cell localization was observed only in the spleen in just one animal. Labeled cells were identified in bone marrow aspirates from three animals following injection, suggesting a role for bone marrow engraftment and repopulation as a possible mechanism for progenitor cell localization in myocardium.

Direct comparison of umbilical cord blood versus bone marrow-derived endothelial precursor cells in mediating neovascularization in response to vascular ischemia.

Endothelial precursor cells (EPCs) cultured from adult bone marrow (BM) have been shown to mediate neovasculogenesis in murine models of vascular injury. We sought to directly compare umbilical cord blood (UCB)- and BM-derived EPC surface phenotypes and in vivo functional capacity. UCB and BM EPCs derived from mononuclear cells (MNC) were phenotyped by surface staining for expression of stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and vascular endothelial [VE]-cadherin), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry. The nonobese diabetic/severe combined immunodeficiency murine model of hind-limb ischemia was used to analyze the potential of MNCs and culture-derived EPCs from UCB and BM to mediate neovasculogenesis. Histologic evaluation of the in vivo studies included capillary density as a measure of neovascularization. Surface CXCR4 expression was notably higher on UCB-derived EPCs (64.29%+/-7.41%) compared with BM (19.69%+/-5.49%; P=.021). Although the 2 sources of EPCs were comparable in expression of endothelial and monocyte markers, BM-derived EPCs contained higher proportions of cells expressing stromal cell markers (CD105 and CD73). Injection of UCB- or BM-derived EPCs resulted in significantly improved perfusion as measured by laser Doppler imaging at days 7 and 14 after femoral artery ligation in nonobese diabetic/severe combined immunodeficiency mice compared with controls (P<.05). Injection of uncultured MNCs from BM or UCB showed no significant difference from control mice (P=.119; P=.177). Tissue samples harvested from the lower calf muscle at day 28 demonstrated increased capillary densities in mice receiving BM- or UCB-derived EPCs. In conclusion, we found that UCB and BM-derived EPCs differ in CXCR4 expression and stromal surface markers but mediate equivalent neovasculogenesis in vivo as measured by Doppler flow and histologic analyses.

Evaluation of the reactivity of apoptosis markers before and after cryopreservation in cord blood CD34(+) cells.

Umbilical cord blood (CB) CD34(+) cells, on the basis of flow cytometry analysis, are comprised of multiple populations. In in vitro assays, only CD34(regular) FSC(high) cells are functional and low percentages of nonfunctional CD34(regular) FSC(low) cells were determined to be present in liquid-stored CB. Liquid-stored CD34(regular) FSC(high) cells prior to cryopreservation were judged to be functional by the formation of erythroid and myeloid colonies and transmigration assays. We have further evaluated the occurrence of apoptosis in CB CD34(+) cells using various apoptotic markers to understand better the influence of storage conditions that could be utilized with transplantation of CB. Of the CD34(regular) FSC(low) cells shown in the present study, 20-45% were labeled with the apoptotic reagents annexin-V, fluorescent caspase peptide substrates, and the anti-mitochondrial antibody APO2.7, but these cells were minimally stained with 7-aminoactinomycin-D (7-AAD). These apoptotic reagents identify different cellular targets, indicating the initiation of the apoptotic cascade prior to cryopreservation/thawing. Following cryopreservation and thawing, the apoptotic markers SYTO-16, tetramethyl rhodamine ethyl ester (TMRE), and 7-AAD showed the presence of apoptotic cells. After cryopreservation/thawing, enumeration of CB CD34(+) cells was reduced 10-65% when excluding cells positive for apoptotic markers. We attempted to limit the progression of apoptosis observed after cryopreservation/thawing by the addition of anti-apoptotic reagents z-VAD-fmk (100 microM) and Q-VD-OPH (100 microM) (peptide inhibitors of caspases) without or with the inclusion of survival reagents for CD34(+) cells-stromal-derived factor-1 (SDF-1), stem cell factor (SCF), thrombopoietin, and diprotin A, an inhibitor of CD26 prior to cryopreservation. The expression of apoptosis markers was minimally affected even when using combinations of caspase inhibitors/ CD34(+) cell survival cytokines in an attempt to block apoptosis caused by cryopreservation/thawing. Decreases in apoptosis marker reactivity following cryopreservation were not observed except for a reduced expression of APO2.7 reactivity with z-VAD-fmk and Q-VD-OPH caspase inhibitors. The ability of the inhibitors of apoptosis of CD34(+) cells to generate CFU-GM, CFU-MK, or BFUE colonies was also unaffected except with z-VAD-fmk (100 microM) and Q-VD-OPH (100 microM). The occurrence of apoptosis, as measured by flow cytometry with selected apoptotic markers, suggests a reduction in the number of viable CD34(+) cells.

Retention of cellular properties of PBPCs following liquid storage and cryopreservation.

BACKGROUND: G-CSF-mobilized PBPCs are routinely cryopreserved within 24 hours of collection. The ability to hold PBPCs for extended time would offer increased flexibility for patients and hospitals. Retention of PBPC properties following overnight shipping, extended liquid storage at 1 to 6 degrees C, and cryopreservation was evaluated. STUDY DESIGN AND METHODS: PBPCs were stored in liquid at 1 to 6 degrees C up to 3 days, with and without shipping, and then cryopreserved in HES (6%), DMSO (5%), and HSA (4%). Thawed samples were assayed after two procedures, on dilution and after dilution and washing. Nucleated cells, viability, CD34+ cell number, committed progenitor colonies, and long-term culture-initiating cells were measured. RESULTS: CD34+ cell number, committed colony-forming cells, and long-term culture-initiating cells were essentially maintained when samples were stored in liquid for 1, 2, or 3 days before cryopreservation or after thawing and dilution. Nevertheless, significant (p < 0.05, paired t test) losses in total nucleated cell numbers were observed if thawed PBPC samples were washed before assay. CONCLUSION: PBPCs can be maintained at 1 to 6 degrees C for up to 3 days and can be cryopreserved after extended storage with properties minimally altered. Dilution alone, without centrifugation and washing, of thawed PBPC samples is a satisfactory procedure for preparing samples for in vitro assays.

Increased transmigration of G-CSF-mobilized peripheral blood CD34+ cells after overnight storage at 37 degrees C.

BACKGROUND: G-CSF-mobilized PBPCs are utilized in allogeneic and autologous PBPC transplants. Homing, adhesion, and transmigration of hematopoietic CD34+ cells are required for successful engraftment. Hematopoietic CD34+ cells undergo directional migration toward the CXCR4 receptor ligand stromal-derived factor-1 (SDF-1). Limited data are available on the effects of liquid storage and cryopreservation on PBPC CD34+ cells. STUDY DESIGN AND METHODS: Magnetic-assisted cell sorting (MACS)-selected CD34+ cells were assayed for retention of in vitro transmigration and phenotypic changes of unit-matched liquid-stored and cryopreserved PBPC samples from healthy donors. Studies evaluated whether transmigration of CD34+ cells in Iscove's modified Dulbecco's medium plus 1 percent HSA alone or in medium supplemented with SCF or allogeneic plasma was affected by overnight incubation at 37 degrees C, relative to nonincubated conditions. RESULTS: Transmigration was maintained during liquid storage at 1 to 6 degrees C during a 2-day period and in unit-matched cryopreserved-thawed samples that had been initially liquid stored. Overnight incubation at 37 degrees C of MACS-selected unit-matched liquid-stored or cryopreserved-thawed CD34+ cells resulted in substantially increased transmigration, in particular with noncoated filters chemoattracted with the chemokine SDF-1. CONCLUSION: CD34+ cell transmigration was comparable between liquid-stored and cryopreserved samples, and both demonstrated similar increases after overnight incubation at 37 degrees C.

Characterization of multiple CD34+ cell populations in cord blood.

Unlike granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood, which show a single homogeneous population of CD34(+) cells, umbilical cord blood (CB) CD34(+) cells are present as multiple populations, CD34(regular) and CD34(bright) (the latter comprising 7.0-58.2% of the total CD34(+) cells), using the ProCOUNT trade mark procedure or with anti-CD34 labeling of immunoselected cells. The CD34(regular) population contains cells with high forward scatter (CD34(regular)FSC(high)) and with low forward scatter (CD34(regular) FSC(low)). Immunomagnetically selected CD34(+) cells, sorted into CD34(regular), CD34(regular) FSC(high), CD34(regular)FSC(low), and CD34(bright) cell populations, were used in in vitro assays: only the CD34(regular)FSC(high) population transmigrated and showed growth of colony-forming unit (CFU) and long-term culture initiating cells (LTC-IC) colonies. The absolute number of CD34(+) cells in CB samples was determined by ProCOUNT trade mark and Stem Kit trade mark enumeration protocols. In liquid stored CB units, ProCOUNT trade mark and Stem Kit trade mark count differences are accounted for by the enumeration of CD34(bright) cells. Differences between ProCOUNT trade mark and Stem Kit trade mark counts using cryopreserved/thawed samples are accounted for by increased CD34(regular) FSC(low) cell numbers (2.0 +/- 1.4% in liquid stored and 27.8 +/- 14.6% in cryopreserved/thawed samples). The ProCOUNT trade mark assay includes the nonfunctional CD34(bright) and CD34(regular)FSC(low) cells as part of the CD34(+) cell count, thereby elevating the absolute number of CD34(+) cells. Using the Stem Kit trade mark assay method of gating, CD34(bright) and CD34(regular)FSC(low) cells are not counted. Our data indicate that the CD34(regular)FSC(high) cell population has functional characteristics based on the in vitro assays and a more accurate count of these cells can be achieved using the Stem Kit trade mark assay.

Weak platelet agonists and U46619 induce apoptosis-like events in platelets, in the absence of phosphatidylserine exposure.

Platelets express apoptotic markers during storage, while aging and after stimulation with strong agonists thrombin and collagen. It is unknown if the weak agonists ADP and epinephrine or U46619, a thromboxane analog, induce the expression of apoptotic markers in platelets. To answer this question, we measured phosphatidylserine exposure, gelsolin cleavage and decrease in membrane mitochondrial potential after stimulation with these agonists. No phosphatidylserine exposure was evident, however, gelsolin cleavage and a platelet population with a decreased membrane mitochondrial potential appeared, suggesting that in platelets selective agonists can induce apoptosis in the absence of phosphatidylserine exposure. Interestingly, costimulation by thrombin plus collagen together with each of the other agonists increased the phosphatidylserine exposure induced by strong agonists. These findings may be of importance in platelet activation and apoptosis under pathophysiological conditions where multiple effectors are involved.