Chondrocytes, Mesenchymal Stem Cells, and Their Combination in Articular Cartilage Regenerative Medicine.

Articular cartilage (AC) is a highly organized connective tissue lining, covering the ends of bones within articulating joints. Its highly ordered structure is essential for stable motion and provides a frictionless surface easing load transfer. AC is vulnerable to lesions and, because it is aneural and avascular, it has limited self-repair potential which often leads to osteoarthritis. To date, no fully successful treatment for osteoarthritis has been reported. Thus, the development of innovative therapeutic approaches is desperately needed. Autologous chondrocyte implantation, the only cell-based surgical intervention approved in the United States for treating cartilage defects, has limitations because of de-differentiation of articular chondrocytes (AChs) upon in vitro expansion. De-differentiation can be abated if initial populations of AChs are co-cultured with mesenchymal stem cells (MSCs), which not only undergo chondrogenesis themselves but also support chondrocyte vitality. In this review we summarize studies utilizing AChs, non-AChs, and MSCs and compare associated outcomes. Moreover, a comprehensive set of recent human studies using chondrocytes to direct MSC differentiation, MSCs to support chondrocyte re-differentiation and proliferation in co-culture environments, and exploratory animal intra- and inter-species studies are systematically reviewed and discussed in an innovative manner allowing side-by-side comparisons of protocols and outcomes. Finally, a comprehensive set of recommendations are made for future studies.

Real-time analysis of immunogen complex reaction kinetics using surface plasmon resonance.

Real-time biospecific interactions of immunogens, measured via BIAcore, were used to verify qualitatively a biosensor design which relies on analyte binding competition reactions to open cross-linked receptor channels. The complexes of importance are: (1) cardiac troponin I (TnI) and monoclonal mouse anti-TnI IgG mAb 265, (2) TnI and bispecific antibodies (BsAbs) which on one end recognize TnI while the other end recognizes nicotinic acetylcholine receptors (nAChRs), (3) nAChRs and rat anti-nAChR IgG mAb 148, (4) nAChRs and BsAbs, (5) nAChRs and Fab'148-TnI biopolymers, and (6) mAb 265 and Fab-TnI biopolymers. A commonly used sensor chip, CM5, was employed to immobilize TnI by covalent amine coupling, while bilayer membrane-associated protein, nAChR, was noncovalently sequestered on a HPA sensor chip via hydrophobic adsorption of membrane lipids. The epitopes of membrane-bound nAChRs were still available to immunogens after being immobilized. Kinetic rate constants and affinities of these systems were calculated from BIAcore sensorgrams. The order of magnitude for dissociation rate constants of the BsAb/TnI linker complex and biopolymer/mAb 265 complex is 10(-2) s-1, which provides an opportunity for competitive binding of free analyte in the sensing systems.

Preparation and characterization of bifunctional biopolymers for receptor-based liposomal immunosensing.

In this study, we prepared bifunctional biopolymers for development of a novel liposomal immunosensing element. These biopolymers were produced such that a rat monoclonal antibody fragment Fab' was linked to a cardiac protein Troponin I (TnI) peptide by a cross-linking reagent, o-phenylenedimaleimide (o-PDM) or N-sucinimidyl 3-(2-pyridyldithio)propionate (SPDP). The biopolymer formation yields were approximately 10% for Fab-TnIMal and 30% for Fab-TnISPDP. Molar ratios of Fab' to SPDP or o-PDM and conjugated Fab' to TnI peptide and conjugation pH have considerable effects on the biopolymer yield. Purification of these biopolymers was achieved by employing size-exclusion HPLC. These biopolymers can bind to receptor channels on one end, while the peptide end can be recognized by an anti-TnI antibody serving as a protein linker to block the channels in the immunosensing element. Then reactions may be used where free analyte competes for cross-linker binding sites whereby channels are rendered active. Characterization of purified biopolymers was performed using gel electrophoresis, ELISAs, and a BIAcore instrument. Furthermore, results of real-time biospecific interaction experiments with use of the BIAcore show that competition binding reactions of free TnI peptide occurred in this new immunosensing design. The binding activities of these two biopolymers are slightly different.

Kinetic modeling and analysis of a vesicle system for immunosensor development.

A novel mechanism is presented for immunosensor development that uses an immunological competition reaction in a vesicle system. This system consists of a suspension of reconstituted vesicles, channel agonist, protein linker to block the channels, voltage sensitive dye and analyte to be detected. In the proposed mechanism analyte serves a catalytic role as individual analytes competitively displace multiple channel linkers through association with one channel, dissociation and new associations with other channels. When one channel opens on a vesicle a permanent Nernst potential develops for that vesicle leading to fluorescence of voltage sensitive dyes. The time constant of the redistribution from linker-channel form to analyte-channel form is 0.92/k4 (k4 is the off-rate constant for the analyte-channel association) in the region of analyte concentrations less than 10(-9) M. Kinetic analyses show that several factors, including concentration of analyte or linker, number of channels per vesicle, on-rate or off-rate constant of the linker-channel and on-rate constant of analyte-channel complexes have significant effects on the minimum signal response time.

Bispecific antibody modification of nicotinic acetylcholine receptors for biosensing.

Recent results show that bispecific antibodies can be used to tailor the selectivity of nicotinic acetylcholine receptors for biosensing purposes. The nicotinic acetylcholine receptors reconstituted in bilayer lipid membranes are inactivated when two bispecific antibodies, attached to the same receptor, bind to a single antigen molecule. Experiments with patch clamp recording equipment reveal that antigen levels of 10(-8) M completely and irreversibly inactivate small numbers of nicotinic acetylcholine receptors. This approach may lead to the construction of biosensors capable of detecting individual antibody-antigen (Ab-Ag) binding events.

A highly stable and selective biosensor using modified nicotinic acetylcholine receptor (nAChR).

Methods for developing stable, sensitive and selective bilayer lipid membrane (BLM)-based biosensors are discussed. Stable BLMs were formed over micromachined polyimide apertures. Selective sensors were made by incorporating nicotinic acetylcholine receptors (nAChRs) modified with bispecific antibodies (BsAbs). When two BsAbs, attached to one nAChR, encounter antigen (Ag), channels are blocked. Sensitivity to single Ag molecules would be possible by monitoring closure of individual nAChRs.

Efficient optimization of ELISAs.

In this paper, we describe a technique for using the statistical method of fractional factorial design in the optimization of an ELISA. Fractional factorial design dramatically reduces the total number of experiments required in the optimization. In addition, this technique enables us to determine the parameters that give the maximum sensitivity range with the most accuracy for the ELISA studied.

Application of compiled BASIC in developing software for collection and analysis of neuronal firing frequency data.

Two programs are described which use an IBM-AT compatible personal computer, equipped with an analog-to-digital converter, to collect and analyze electrophysiological data. The first program is used to determine neuron firing rates during intracellular experiments and to save these results on disk. The second program is used to perform off-line analysis of the frequency data. Both programs are written entirely in the well known BASIC language and the Microsoft QuickBASIC compiler is employed for their use. All of the necessary hardware can be purchased commercially. In this paper emphasis is placed on the strategies and limitations involved when this high-level language is applied to tasks often needed in data acquisition and analysis. Both on- and off-line collection schemes are considered. This software is available from the authors.

Effects of temperature and analyte application technique on neuron-based chemical sensing.

Results are presented which enhance the field of neuron-based sensing by providing insight on the effects of operating temperature and analyte application technique (pulse versus back-mixed) on sensing properties. In these studies, serotonin sensing attributes of giant visceral neurons VV1 and VV2 from the pond snail Lymnea stagnalis were measured. Experiments using a rapid fluid-exchange system reveal a concentration-dependent increase in maximum firing frequency similar to that reported earlier for a slow well-mixed application. With a rapid application, however, the maximum firing frequency is reached more quickly, and there is less cell-to-cell variability in both the maximum response and sensitivity. Given an application technique, an increase in temperature causes an increase in sensitivity and maximum firing frequency, as well as a decrease in the time required for the response to return to baseline following removal of the analyte. To provide insights on the kinetics of the serotonin-induced response, the effects of temperature and concentration on the rates of activation, recovery and desensitization were examined in detail. In general, it was found that an increase in temperature increases the rates of activation and desensitization, while the effects on recovery were not apparent. In addition, both the rates of activation and desensitization have a direct dependence on concentration while the rate of recovery has an inverse dependence.

A novel continuous centrifugal bioreactor for high-density cultivation of mammalian and microbial cells.

A continuous centrifugal bioreactor (CCBR), developed to study the growth and productivity of dense suspensions cultures, has been applied to both fermentation and mammalian cell cultivation processes. With this approach, high-density nonflocculent cultures are maintained in a tapered fluidized bed by balancing the drag forces on the cells due to following substrate with the centrifugal forces. The Sysyem was first used to produce ethanol by fermentation with Saccharomyces cerevisiae; then with H21A1 mouse hybridoma cells secreting monoclonal antibody (MoAb), lgM. Results of this research show the feasibility of using the CCBR for both production of secreted products and as a research tool for studying cell metabolism and production kinetics. Media recycle may be used to modify the behavior of the system form a plug flow apparatus to a continuous stirred reactor (CSTR).

Optimization of glass microelectrode properties by response surface methodology.

Glass microelectrodes filled with electrolyte solutions are standard tools for electrophysiological studies. However, for any given application, there are limitations to the properties of the microelectrode, such as impedance and shank length, that can yield satisfactory results. The trial and error approach in pulling electrodes with the desired properties can be time consuming. The use of a response surface procedure which allows the experimenter to change more than one factor at a time and therefore determine the desired puller condition more efficiently is demonstrated. Also, design improvements for the World Precision Instrument, Model PUL-1, Microelectrode puller, used in this study are suggested.

Control and optimization of apheresis procedures in a COBE 2997 cell separator.

To obtain more efficient operation of a COBE Model 2997 clinical cell separator using either a Single Stage II (SS II) or a Dual Stage separation chamber, modifications were made to allow complete computer control. Product cell density was detected using an optical sensor and controlled by automatic feedback through a microcomputer interface. Control was accomplished by automatically adjusting the red blood cell (RBC) and plasma product flow rates using a proportional-integral (PI) algorithm. Results show that, using either chamber, the product cell density can be maintained at a preselected value for extended periods of time without operator intervention. This system allowed investigation of optimal operating regions for plateletpheresis and leukapheresis procedures. The effects of centrifuge rpm and controller set point on centrifuge operation were investigated using a second order factorial experimental design. Theoretical significance of model parameters was assessed with the aid of a hindered settling model and simple reasoning about the interface position relative to the collection port. The results suggest that, in either chamber, the optimum operating region for plateletpheresis procedures occurs at moderate controller set points and high centrifuge rpm. The resultant operating efficiency and product purity values are approximately 63 percent and 0.65 respectively in the SS II chamber and approximately 70 percent and 0.70 respectively in the Dual Chamber. In the SS II, the optimum operating region for leukapheresis procedures occurred at high controller set point values for any centrifuge rpm above 1200 with an operating efficiency near 100 percent. However, in the Dual Chamber, the optimum operating region for leukapheresis procedures occurred at high controller set points and high centrifuge rpm's, again providing an operating efficiency near 100 percent.

Evaluation of neuron-based sensing with the neurotransmitter serotonin.

Results are presented on the development of a novel biosensor which will use neurons or neuronal components as both the recognition elements and primary transducers for analyte quantitation. This concept is demonstrated and evaluated by exposing identified neurons from the visceral ganglia of the pond snail Limnea stagnalis to the model analyte serotonin. Experiments reveal a reversible, concentration-dependent increase in the rate of spontaneous action potential generation, over a concentration range of four orders of magnitude. Studies with the antagonist methysergide verify that this response is mediated through serotonin-sensitive receptors. Exposure of the neurons to serotonin causes the firing frequency to rapidly increase to a maximum and then slowly diminish to a sub-optimal level. It was found that the maximum frequency provides an indication of chemical concentration that is repeatable. Data are also presented which further advance the field of neuronal biosensing by demonstrating both the effects of cell to cell variability on response reproducibility and the effects of the desensitizing response on the operation of a neuron-based sensor in both a continuous and discontinuous mode.

Interface dynamics in a centrifugal cell separator.

Experiments were performed with bovine blood to study the response of the plasma/cell interface position to pumping rate adjustments in two single-stage (SS I and SS II) cell separation chambers. Standard clinical instrumentation and equipment and on-line computer interfacing were used to monitor and control the interface position. The data provided a quantitative description of its dynamics in the SS I and the SS II chambers. In the SS I chamber, adjustments in the flow rate caused the interface to move very slowly and in a complex manner from one steady-state operation position to another. Such behavior made both manual and computer-controlled operation very difficult. By contrast, the SS II chamber was inherently unstable for most operating conditions. We demonstrated, however, that a feedback controller could be used easily to adjust or maintain the interface position, and this system moved from one steady-state operating condition to another 10 times as fast as the system using the SS I chamber. Also, the manner in which the controller allowed the system to respond to operator requests was much simplier than that for the SS I system.

Particle interaction effects on blood cell sedimentation and separations.

Experiments with a continuous centrifuge reveal that the separation of leukocytes (WBCs) and platelets from erythrocytes (RBCs) is maximized at a lower than normal RBC concentration or hematocrit (HCT). Conventional hindered settling models are unsatisfactory in the explanation of this behavior because they do not adequately account for differences in cell size, density, deformability and electrical charge on the membrane surface. In this paper a new approach is taken where an effective porosity is used to account for the differences in the micro-environment of each particle type. Introduction of effective porosities into various sedimentation equations is useful in allowing better prediction of the optimum separation conditions observed in experimental data. Further adjustment of parameters is necessary for some models to shift optimums in settling curves to align with experimental trends.

The effects of RPM and recycle on separation efficiency in a clinical blood cell centrifuge.

A COBE blood cell centrifuge, model 2997 with a single stage channel, was modified to allow computer controlled sampling, and to allow recycle of red blood cells (RBCs) and plasma streams using bovine whole blood. The effects of recycle of the packed RBC and plasma product streams, and of the centrifuge RPM on platelet and white blood cell (WBC) separation efficiencies were quantified using a central composite factorial experimental design. These data were then fit using second order models. Both the model for the WBC separation efficiency and the model for the platelet separation efficiency predict that RPM has the greatest effect on separation efficiency and that RBC and plasma recycle have detrimental effects at moderate to low RPM, but have negligible impact on separation efficiency at high RPM.

Experimental considerations for the centrifugal separation of blood cell components.

Processing techniques using recycle and staging for blood cell collection lead to numerous conceptual design configurations. From a practical viewpoint, many of these schemes can become incongruous. Experimental fractional recoveries with up to six stages are presented, and material balance considerations and process comparisons are made. Isolation of specific leukocyte types, and cell viability effects are studied.

The effect of recycle on the continuous centrifugal processing of blood cells.

Recycle configurations are presented for continuous-flow blood cell separation systems. A sample design calculation is presented for a single-stage unit with recycle. Increases in blood-cell collection efficiencies may be obtained with recycle, and various design configurations are proposed.