Impact of allogeneic feline uterine-derived mesenchymal stromal cell intravenous treatment on renal function of nephrectomized cats with chronic kidney disease.

Chronic kidney disease (CKD) is a common condition and leading cause of mortality in cats. Mesenchymal stromal cells (MSCs) may have a therapeutic effect on CKD. The aim of this pilot study was to determine efficacy of systemically-administered allogeneic uterine tissue-derived MSCs (UMSCs) in cats with CKD. Eighteen renal-compromised, unilaterally nephrectomized cats received two doses of 3 × 107 allogeneic UMSCs given intravenously (IV) with a 2-week dose interval. The primary endpoint was renal function, with treatment success defined by a 20% increase in glomerular filtration rate (GFR; iohexol clearance) and/or a 20% decrease in plasma creatinine in 50% of the cats. Secondary endpoints included diet and water consumption, body weight, urine characteristics, and adverse events. Treatment was well tolerated and associated with a statistically meaningful increase in GFR on Days 13, 28, 57, 99, 121 and 182, compared with baseline (P < 0.0001 for Days 13 to 99 inclusive; P = 0.0029 and P = 0.0225 for Days 121 and 182, respectively). Greater than 50% of the cats demonstrated a 20% increase in GFR on all days except Day 150, at which point GFR measurements were consistently above baseline. Statistically meaningful increases in diet and water consumption were observed. Substantial improvements in GFR were observed throughout the six-month evaluation period (excluding Day 150) in more than 50% of cats, thereby meeting the primary endpoint. Therefore, this IV-administered, allogeneic cellular therapy may support both renal function and clinical status of cats with CKD.

Autologous bone marrow concentrate intradiscal injection for the treatment of degenerative disc disease with three-year follow-up.

PURPOSE: The purpose of this study is to assess safety and feasibility of intradiscal bone marrow concentrate (BMC) injections to treat low back discogenic pain as an alternative to surgery with three year minimum follow-up. METHODS: A total of 26 patients suffering from degenerative disc disease and candidates for spinal fusion or total disc replacement surgery were injected with 2 ml autologous BMC into the nucleus pulposus of treated lumbar discs. A sample aliquot of BMC was characterized by flow cytometry and CFU-F assay to determine progenitor cell content. Improvement in pain and disability scores and 12 month post-injection MRI were compared to patient demographics and BMC cellularity. RESULTS: After 36 months, only six patients progressed to surgery. The remaining 20 patients reported average ODI and VAS improvements from 56.7 ± 3.6 and 82.1 ± 2.6 at baseline to 17.5 ± 3.2 and 21.9 ± 4.4 after 36 months, respectively. One year MRI indicated 40% of patients improved one modified Pfirrmann grade and no patient worsened radiographically. Cellular analysis showed an average of 121 million total nucleated cells per ml, average CFU-F of 2713 per ml, and average CD34+ of 1.82 million per ml in the BMC. Patients with greater concentrations of CFU-F (>2000 per ml) and CD34+ cells (>2 million per ml) in BMC tended to have significantly better clinical improvement. CONCLUSIONS: There were no adverse events related to marrow aspiration or injection, and this study provides evidence of safety and feasibility of intradiscal BMC therapy. Patient improvement and satisfaction with this surgical alternative supports further study of the therapy.

Percutaneous injection of autologous bone marrow concentrate cells significantly reduces lumbar discogenic pain through 12 months.

Degenerative disc disease (DDD) induces chronic back pain with limited nonsurgical options. In this open label pilot study, 26 patients (median age 40 years; range 18-61) received autologous bone marrow concentrate (BMC) disc injections (13 one level, 13 two levels). Pretreatment Oswestry disability index (ODI) and visual analog scale (VAS) were performed to establish baseline pain scores (average 56.5 and 79.3, respectively), while magnetic resonance imaging was independently scored according to the modified Pfirrmann scale. Approximately 1 ml of BMC was analyzed for total nucleated cell (TNC) content, colony-forming unit-fibroblast (CFU-F) frequency, differentiation potential, and phenotype characterization. The average ODI and VAS scores were reduced to 22.8 and 29.2 at 3 months, 24.4 and 26.3 at 6 months, and 25.0 and 33.2 at 12 months, respectively (p ≤ .0001). Eight of twenty patients improved by one modified Pfirrmann grade at 1 year. The average BMC contained 121 × 10(6) TNC/ml with 2,713 CFU-F/ml (synonymous with mesenchymal stem cells). Although all subjects presented a substantial reduction in pain, patients receiving greater than 2,000 CFU-F/ml experienced a significantly faster and greater reduction in ODI and VAS. Subjects older than 40 years who received fewer than 2,000 CFU-F/ml experienced an average pain reduction of 33.7% (ODI) and 29.1% (VAS) at 12 months, while all other patients' average reduction was 69.5% (ODI, p = .03) and 70.6% (VAS, p = .01). This study provides evidence of safety and feasibility in the nonsurgical treatment of DDD with autologous BMC and indicates an effect of mesenchymal cell concentration on discogenic pain reduction.

Short Term Culture of Human Mesenchymal Stem Cells with Commercial Osteoconductive Carriers Provides Unique Insights into Biocompatibility.

For spinal fusions and the treatment of non-union fractures, biological substrates, scaffolds, or carriers often are applied as a graft to support regeneration of bone. The selection of an appropriate material critically influences cellular function and, ultimately, patient outcomes. Human bone marrow mesenchymal stem cells (BMSCs) are regarded as a critical component of bone healing. However, the interactions of BMSCs and commercial bone matrices are poorly reported. BMSCs were cultured with several commercially available bone substrates (allograft, demineralized bone matrix (DBM), collagen, and various forms of calcium phosphates) for 48 h to understand their response to graft materials during surgical preparation and the first days following implantation (cell retention, gene expression, pH). At 30 and 60 min, bone chips and inorganic substrates supported significantly more cell retention than other materials, while collagen-containing materials became soluble and lost their structure. At 48 h, cells bound to ß-tricalcium phosphate-hydroxyapatite (ßTCP-HA) and porous hydroxyapatite (HA) granules exhibited osteogenic gene expression statistically similar to bone chips. Through 24 h, the DBM strip and ßTCP-collagen became mildly acidic (pH 7.1-7.3), while the DBM poloxamer-putties demonstrated acidity (pH < 5) and the bioglass-containing carrier became basic (pH > 10). The dissolution of DBM and collagen led to a loss of cells, while excessive pH changes potentially diminish cell viability and metabolism. Extracts from DBM-poloxamers induced osteogenic gene expression at 48 h. This study highlights the role that biochemical and structural properties of biomaterials play in cellular function, potentially enhancing or diminishing the efficacy of the overall therapy.

MSC frequency correlates with blood vessel density in equine adipose tissue.

Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to develop into different mature mesenchymal cell types. They were originally isolated from bone marrow, but MSC-like cells have also been isolated from other tissues. The common feature of all of these tissues is that they all house blood vessels. It is, thus, possible that MSCs are associated with perivascular locations. The objective of this work was to test the hypothesis that MSCs are associated with blood vessels by verifying if MSC frequency positively correlates with blood vessel density. To this end, samples from highly and poorly vascularized adipose tissue sites of two equine donors were collected and processed for histology and cell isolation. MSC frequency in these samples was estimated by means of CFU-F assays, which were performed under MSC conditions. Culture-adherent cells from equine adipose tissue and bone marrow were culture expanded, tested for differentiation into mesenchymal cell types in vitro, and implanted in vivo in porous ceramic vehicles to assess their osteogenic capacity, using human MSCs and brain pericytes as controls. The differentiation assays showed a difference between adipose tissue-derived cells as compared to equine bone marrow MSCs. While differences in CFU-F frequencies between both donors were evident, the CFU-F numbers correlated directly with blood vessel densities (r(2) = 0.86). We consider these preliminary data as further evidence linking MSCs to blood vessels.

Development of an immunosensor based on pressure transduction.

Traditional strategies for signal transduction in immunosensors are based on piezoelectric, thermometric, electrochemical, magnetic and optical methods. The use of pressure as a signal transduction method in immunosensors has not been reported previously. An immunosensor incorporating the detection of a change in pressure as the signal-transducing mechanism was investigated. A commercially available ultra-low pressure sensor was used in conjunction with a sealed chamber to assess the feasibility of this strategy. A key feature of the current approach is the use of a thin membrane (or film) in which to perform an immunoassay and subsequently to detect production of gas. The thinness contributes to efficient gas evolution and minimizes the effect of liquid acting as a "sink" for gas molecules. This feature also simplifies measurement of evolved gas, which traditionally was based on the use of bulk solutions, shaking and pH changes to "release" dissolved gas (especially carbon dioxide). Gas generation in the current approach is achieved by the coupling of catalase to haptens or antibodies for use in competitive or sandwich immunoassays, respectively. Hydrogen peroxide is used as the substrate. Performance characteristics of the sensor apparatus were assessed in several ways. Injection of various volumes of air from a gas-tight syringe produced an essentially linear relationship from 0.2 to 2.0 microl of injected volume, with a slope of approximately 5 V/microl. Depending on the duration of the sampling period, specific signals in excess of 2 V have been obtained for 0.01 units of catalase (approximately 0.4 ng of protein). Development and use of this sensing apparatus will be described for both competitive and sandwich immunoassays.