Human mesenchymal stromal cells attenuate graft-versus-host disease and maintain graft-versus-leukemia activity following experimental allogeneic bone marrow transplantation.

We sought to define the effects and underlying mechanisms of human, marrow-derived mesenchymal stromal cells (hMSCs) on graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) activity. Irradiated B6D2F1 mice given C57BL/6 BM and splenic T cells and treated with hMSCs had reduced systemic GvHD, donor T-cell expansion, and serum TNFα and IFNγ levels. Bioluminescence imaging demonstrated that hMSCs redistributed from lungs to abdominal organs within 72 hours, and target tissues harvested from hMSC-treated allogeneic BMT (alloBMT) mice had less GvHD than untreated controls. Cryoimaging more precisely revealed that hMSCs preferentially distributed to splenic marginal zones and regulated T-cell expansion in the white pulp. Importantly, hMSCs had no effect on in vitro cytotoxic T-cell activity and preserved potent GvL effects in vivo. Mixed leukocyte cultures containing hMSCs exhibited decreased T-cell proliferation, reduced TNFα, IFNγ, and IL-10 but increased PGE2 levels. Indomethacin and E-prostanoid 2 (EP2) receptor antagonisms both reversed while EP2 agonism restored hMSC-mediated in vitro T-cell suppression, confirming the role for PGE2 . Furthermore, cyclo-oxygenase inhibition following alloBMT abrogated the protective effects of hMSCs. Together, our data show that hMSCs preserve GvL activity and attenuate GvHD and reveal that hMSC biodistribute to secondary lymphoid organs wherein they attenuate alloreactive T-cell proliferation likely through PGE2 induction.

High-resolution kinetics of cytokine signaling in human CD34/CD117-positive cells in unfractionated bone marrow.

Cytokine-mediated phosphorylation of Erk (pErk), ribosomal S6 (pS6), and Stat5 (pStat5) in CD34(+)/CD117(+) blast cells in normal bone marrow from 9 healthy adult donors were analyzed over 60 minutes. Treatment with stem cell factor (SCF), Flt3-ligand (FL), IL-3, and GM-CSF and measurement by multiparametric flow cytometry yielded distinctive, highly uniform phosphoprotein kinetic profiles despite a diverse sample population. The correlated responses for SCF- and FL-stimulated pErk and pS6 were similar. Half the population phosphorylated Erk in response to SCF between 0.9 and 1.2 minutes, and S6 phosphorylation followed approximately a minute later (t½(pS6 rise) = 2.2-2.7 minutes). The FL response was equally fast but more variable (t½(pErk rise) = 0.9-1.3 minutes; t½(pS6 rise) = 2.5-3.5 minutes). Stat5 was not activated in 97% of the cells by either cytokine. IL-3 and GM-CSF were similar to each other with half of blast cells phosphorylating Stat5 and 15% to 20% responding through Erk and S6. Limited comparison with leukemic blasts confirmed universal abnormal signaling in AML that is significantly different from normal bone marrow blasts. These differences included sustained signals, a larger fraction of responding cells, and amplification of phosphorylation levels for at least one phosphoprotein. These data support the eventual use of this approach for disease diagnosis and monitoring.

Growth factors and craniofacial surgery.

The specialty of craniofacial surgery is broad and includes trauma, aesthetics, reconstruction of congenital deformities, and regeneration of tissues. Moreover, craniofacial surgery deals with a diverse range of tissues including both "soft" and "hard" tissues. Technological advances in materials and biological sciences and improved surgical techniques have remarkably improved clinical outcomes. The quest to raise the bar for patient care continues to inspire advances for predictable biological regeneration of soft and hard tissues. As a consequence of this quest for advancement, a wide spectrum of biologicals is becoming available to surgeons. Is the use of recombinant DNA engineered biologicals daring? Sensible? Logical? Timely? Safe? It is crucial for the practicing craniofacial surgeon to take a step back periodically and carefully review the biological factors that have the potential for dramatically altering the discipline of craniofacial surgery. With this emphasis, the coauthors of this article will focus on growth factor technology underscoring bone tissue regeneration. As the 21st-century matures, recombinant human biologicals will have an overwhelming impact on the practice of craniofacial surgery.

Reciprocal Th1 and Th17 regulation by mesenchymal stem cells: Implication for multiple sclerosis.

Human mesenchymal stem cells (hMSCs) are being considered for clinical trials of multiple sclerosis (MS). We examined the effects of adult bone marrow-derived hMSCs on responses of primary human Th1, Th17, and Th1/17 double-expressing T-cell subsets, all implicated in MS. As expected, soluble products from hMSCs inhibited Th1 responses; however, Th17 responses were increased. Secretion of interleukin (IL)-10, considered anti-inflammatory, was decreased. Pretreating hMSCs with the proinflammatory cytokine IL-1ß accentuated these effects, and caused decreases in the Th1/17 subset. These findings underscore the importance of further preclinical work and immune-monitoring to define hMSC effects on disease-relevant immune responses under variable conditions.

Regenerative stromal cell therapy in allogeneic hematopoietic stem cell transplantation: current impact and future directions.

Regenerative stromal cell therapy (RSCT) has the potential to become a novel therapy for preventing and treating acute graft-versus-host disease (GVHD) in the allogeneic hematopoietic stem cell transplant (HSCT) recipient. However, enthusiasm for using RSCT in allogeneic HSCT has been tempered by limited clinical data and poorly defined in vivo mechanisms of action. As a result, the full clinical potential of RSCT in supporting hematopoietic reconstitution and as treatment for GVHD remains to be determined. This manuscript reviews the immunomodulatory activity of regenerative stromal cells in preclinical models of allogeneic HSCT, and emphasizes an emerging literature suggesting that microenvironment influences RSC activation and function. Understanding this key finding may ultimately define the proper niche for RSCT in allogeneic HSCT. In particular, mechanistic studies are needed to delineate the in vivo effects of RSCT in response to inflammation and injury associated with allogeneic HSCT, and to define the relevant sites of RSC interaction with immune cells in the transplant recipient. Furthermore, development of in vivo imaging technology to correlate biodistribution patterns, desired RSC effect, and clinical outcome will be crucial to establishing dose-response effects and minimal biologic dose thresholds needed to advance translational treatment strategies for complications like GVHD.

Transcriptional profiling of human mesenchymal stem cells transduced with reporter genes for imaging.

Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSC transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild-type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and antiapoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic, or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to lose their basic stem cell identity as demonstrated by osteogenic, chondrogenic, and adipogenic differentiation assays with both transduced and wild-type MSCs, although a certain degree of alterations occurred. Histological analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cells while enabling noninvasive imaging in living animals to study the biodistribution and other biological activities of the cells.

Long-lasting multiagent chemotherapy in adult high-risk Ewing's sarcoma of bone.

The outcome of Ewing's sarcoma depends on the anatomical site of the tumor. Studies conducted in high-risk patients are limited. We evaluated the outcome of high-risk Ewing's sarcoma patients that received long-term treatment protocol. Twenty-five patients (22 males, 3 females) with poor prognostic features were treated according to long-term Ewing's sarcoma protocol. Central-axis localization, inadequacy or unavailability of surgical resection, older than 15 years of age, are accepted as high-risk factors. The median age of patients was 23 years (range, 18-55). The tumor localization was pelvis (9), femur (1), tibia (1), fibula (1), maxilla (1), clavicle (1), vertebrae (5), metatarse (1), and ribs (5). Neoadjuvant chemotherapy was applied between weeks 0 and 6, local therapy on week 9, and adjuvant maintenance chemotherapy between weeks 11 and 41. All patients received neoadjuvant and adjuvant maintenance chemotherapy. Local therapy consisted of radiotherapy (32%), surgery alone (12%), or surgery and radiotherapy (56%). The median total treatment period was 10 months. The median follow-up was 25 months (range, 7-89). Three-year cumulative OS and DFS rates were 43% (95% CI, 28.5-57.85) and 40% (95% CI 23.63-52.19), respectively. The most common grade III/IV toxicities observed during the treatment protocol were neutropenia (16%) and gastrointestinal toxicities (16%). Our study indicated that long-term multiagent combination chemotherapy may result in better outcome in adult high-risk patients undergoing adequate surgical resection of the tumor and local radiotherapy. Further randomized studies are needed to assess the efficacy of this treatment protocol in patients with adequate surgical margins.

Reduced dose of lenograstim is as efficacious as standard dose of filgrastim for peripheral blood stem cell mobilization and transplantation: a randomized study in patients undergoing autologous peripheral stem cell transplantation.

In vitro studies have demonstrated a 27% increased efficacy of lenograstim over filgrastim. However, equal doses of 10 microg/kg/day of filgrastim and lenograstim have been recommended for mobilization of CD34+ cells without associated chemotherapy. In this study, we investigated whether a 25% reduced dose of lenograstim at 7.5 microg/kg/day is equavalent to 10 microg/kg/day filgrastim for autologous peripheral blood stem cell (PBSC) mobilization and transplantation. A total of 40 consecutive patients were randomized to either filgrastim (n = 20) or lenograstim (n = 20). The two cohorts were similar in regard to disease, sex, body weight, body surface area, conditioning regimens, previous chemotherapy cycles and radiotherapy. Each growth factor was administered for 4 consecutive days. The first PBSC apheresis was done on the 5th day. In the posttransplant period, the same G-CSF was given at 5 microg/kg/day until leukocyte engraftment. Successful mobilization was achieved in 95% of patients. Successful mobilization with the first apheresis, was achieved in 10/20 (50%) patients in the filgrastim group versus 9/20 (46%) patients in the lenograstim group. No significant difference was seen in the median number of CD34+cells mobilized, as well as the median number of apheresis, median volume of apheresis, percentage of CD34+ cells, and CD34+ cell number. Leukocyte and platelet engraftments, the number of days requiring G-CSF and parenteral antibiotics, the number of transfusions were similar in both groups in the posttransplant period. Lenograstim 7.5 microg/kg/day is as efficious as filgrastim 10 microg/kg/day for autologous PBSC mobilization and transplantation.

Human and Rat Bone Marrow-Derived Mesenchymal Stem Cells Differ in Their Response to Fibroblast Growth Factor and Platelet-Derived Growth Factor.

Tissue engineering requires large numbers of cells with enhanced differentiation properties. Thus, the effect of expansion conditions must be explored. Human and rat marrow-derived mesenchymal stem cells (hMSCs and rMSCs, respectively) were comparatively culture expanded through seven passages in the presence of either fibroblast growth factor-2 (FGF-2) or platelet-derived growth factor BB (PDGF-BB). Proliferation of both hMSCs and rMSCs was enhanced by FGF-2 and PDGF-BB. Population doubling times for hMSCs were 2.4 days for control and 1.75 and 2.0 days for FGF-2 and PDGF-BB, respectively, and 3.25, 3.06, and 2.95 days for rMSCs. Supplementation with FGF-2 during cell expansion resulted in significantly greater in vivo bone formation for hMSCs. Use of PDGF-BB resulted in greater bone formation than that observed for control conditions, but the differences were only significant for P1. For rMSCs, significant increases in bone formation were noted in either FGF-2 or PDGF-BB expanded cells implanted at P4 or P7, but not for P1. Under in vitro osteogenic stimulation, calcium content was elevated and bone matrix deposition was enhanced for P1 and P7 rMSCs expanded with FGF-2. Although culture conditions, including FBS, were held constant, these observations suggest that medium must be optimized separately for each species of MSCs.

Simplification of aggregate culture of human mesenchymal stem cells as a chondrogenic screening assay.

Aggregate culture provides a three-dimensional (3-D) environment for differentiating or differentiated cells; it is particularly useful to study in vitro chondrogenesis and cartilage biology. We have recently ported this method from a conical tube-based format to a 96-well plate format for the study of mesenchymal stem cell (MSC) chondrogenesis. The microplate format has greatly reduced the workload and materials cost, while maintaining reproducible chondrogenic differentiation. A long-term goal is to fully automate aggregate culture--this requires critically identifying all the indispensable steps of the protocol. Robotic laboratory equipment for manipulating microplate assays are commercially available; however centrifugation steps are difficult to implement automatically. We, therefore, tested whether the centrifugation step can be eliminated, thus significantly streamlining the assay workflow. By comparing aggregates prepared from human bone marrow-derived MSCs (hMSCs) that were formed either through centrifugation or through free sedimentation, we found that both methods produce aggregates with similar formation kinetics, and that there was no perceptible difference in the timing of the appearance of markers of chondrogenesis. Thus, it appears safe to eliminate the centrifugation step from the aggregate culture protocol. This results in significant time and effort savings and paves the way for future full automation of the aggregate assay.

Hydrostatic pressure increases apoptosis in cartilage-constructs produced from human osteoarthritic chondrocytes.

Tissue-engineering is considered a promising avenue for developing human articular cartilage implants that can be employed for resurfacing damaged cartilage in the early stages of osteoarthritis. In the present study, human cartilage-constructs were produced from human osteoarthritic chondrocytes maintained on a scaffold of HYAFFR-11 in perfusion mini-bioreactors or after implantation and recovery from nude or SCID mice after 3 weeks. The human cartilage-construct extracellular matrix reacted positively with anti-Type II collagen monoclonal antibody, but not with anti-Type I or anti-Type X collagen monoclonal antibodies. A significant portion of the cartilage-construct extracellular matrix stained metachromatic with Toluidine blue-O indicative of sulfated-proteoglycan deposition. Cyclic hydrostatic pressure applied for 4 hrs at 5 MPa using a 1 Hertz sinusoidal frequency significantly increased (p < 0.02) the proportion of apoptotic cells in the cartilage-constructs (41% +/- 4.2%; mean +/- SD) compared to control cartilage-constructs (28.5 +/- 8.4%).

A rapid seeding technique for the assembly of large cell/scaffold composite constructs.

These studies address critical technical issues involved in creating human mesenchymal stem cell (hMSC)/ scaffold implants for cartilage repair. These issues include obtaining a high cell density and uniform spatial cell distribution within the scaffold, factors that are critical in the initiation and homogeneity of chondrogenic differentiation. For any given scaffold, the initial seeding influences cell density, retention, and spatial distribution within the scaffold, which eventually will affect the function of the construct. Here, we discuss the development of a vacuum-aided seeding technique for HYAFF -11 sponges which we compared to passive infiltration. Our results show that, under the conditions tested, hMSCs were quantitatively and homogeneously loaded into the scaffolds with 90+% retention rates after 24 h in perfusion culture with no negative effect on cell viability or chondrogenic potential. The retention rates of the vacuum-seeded constructs were at least 2 times greater than those of passively seeded constructs at 72 h. Histomorphometric analysis revealed that the core of the vacuum-seeded constructs contained 240% more cells than the core of passively infiltrated scaffolds. The vacuum seeding technique is safe, rapid, reproducible, and results in controlled quantitative cell loading, high retention, and uniform distribution.

High-throughput aggregate culture system to assess the chondrogenic potential of mesenchymal stem cells.

We have developed an improved method for preparing cell aggregates for in vitro chondrogenesis studies. This method is a modification of a previously developed conical tube-based culture system that replaces the original 15-mL polypropylene tubes with 96-well plates. These modifications allow a high-throughput approach to chondrogenic cultures, which reduces both the cost and time to produce chondrogenic aggregates, with no detrimental effects on the histological and histochemical qualities of the aggregates. We prepared aggregates in both systems with human bone marrow-derived mesenchymal stem cells (hMSC). The aggregates were harvested after 2 and 3 weeks in chondrogenic culture and analyzed for their ability to differentiate along the chondrogenic pathway in a defined in vitro environment. Chondrogenic differentiation was assessed biochemically by DNA and glycosaminoglycan (GAG) quantification assays and by histological and immunohistologic assessment. The chondrogenic cultures produced in the 96-well plates appear to be slightly larger in size and contain more DNA and GAG than the aggregates made in tubes. When analyzed histologically, both systems demonstrate morphological characteristics that are consistent with chondrogenic differentiation and cartilaginous extracellular matrix production.

Bioreactors mediate the effectiveness of tissue engineering scaffolds.

We hypothesized that the mechanically active environment present in rotating bioreactors mediates the effectiveness of three-dimensional (3D) scaffolds for cartilage tissue engineering. Cartilaginous constructs were engineered by using bovine calf chondrocytes in conjunction with two scaffold materials (SM) (benzylated hyaluronan and polyglycolic acid); three scaffold structures (SS) (sponge, non-woven mesh, and composite woven/non-woven mesh); and two culture systems (CS) (a bioreactor system and petri dishes). Construct size, composition [cells, glycosaminoglycans (GAG), total collagen, and type-specific collagen mRNA expression and protein levels], and mechanical function (compressive modulus) were assessed, and individual and interactive effects of model system parameters (SM, SS, CS, SM*CS and SS*CS) were demonstrated. The CS affected cell seeding (higher yields of more spatially uniform cells were obtained in bioreactor-grown than dish-grown 3-day constructs) and subsequently affected chondrogenesis (higher cell numbers, wet weights, wet weight GAG fractions, and collagen type II levels were obtained in bioreactor-grown than dish-grown 1-month constructs). In bioreactors, mesh-based scaffolds yielded 1-month constructs with lower type I collagen levels and four-fold higher compressive moduli than corresponding sponge-based scaffolds. The data imply that interactions between bioreactors and 3D tissue engineering scaffolds can be utilized to improve the structure, function, and molecular properties of in vitro-generated cartilage.

Performance of polyoxymethylene plastic (POM) as a component of a tissue engineering bioreactor.

Polyoxymethylene (POM, acetal homopolymer, polyacetal), commercialized as Delrin by DuPont, is an engineering resin with mechanical properties that make it useful for the prototyping and manufacture of laboratory apparatus. These properties include excellent, "metal-like," machining characteristics and dimensional stability, as well as thermal stability, which allows steam sterilization. Historically, POM has been used widely, including as a surgical implant material. For these reasons, we have used this plastic as a media-wetted component in a tissue-engineering bioreactor, with good results. However, a study by LaIuppa et al.5 suggested that POM is unsuitable for use in a cell culture environment (LaIuppa et al. J Biomed Mater Res 1997;36:347-359). POM is based on the polymerization of formaldehyde, and, in addition, contains stabilizers and/or fillers. All of these could potentially be released into the medium, e.g., as formaldehyde or other thermal breakdown products, especially upon repeated autoclaving. The cited report thus appeared plausible, although contrary to our observations. In this study, we specifically assessed whether media conditioned by long-term exposure to machined white POM had a negative effect on the proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs). We selected this cell system, as cartilage tissue engineering is the primary application of our bioreactor system. The POM samples were steam-autoclaved 1 to 20 times, to assess the possibility of any toxic thermal breakdown product release into the media. We found that MSCs did not attach directly to machined POM. Because cells that escape from the tissue construct cannot colonize the reactor and compete for nutrients, this is a desirable characteristic of a material used in a tissue-engineering bioreactor. Furthermore, the use of POM-conditioned media had no detectable impact on the proliferation rate of MSCs measured over a one-week period; nor was any effect on chondrogenic differentiation observed at up to 3 weeks in culture. In summary, the use of POM as a culture medium-wetted component appears to be innocuous, at least for human MSCs. The contrast of these findings to those of LaIuppa et al.5 may reflect a cell-type specific sensitivity, or may be due to different handling of the material.

Repair of osteochondral defect with tissue-engineered two-phase composite material of injectable calcium phosphate and hyaluronan sponge.

Articular cartilage has limited capacity for repair. In the present study, tissue-engineered two-phase composite material was used for the repair of osteochondral defects in young adult rabbit knee. This composite material is composed of an injectable calcium phosphate (ICP) and a hyaluronan (HA) derivate of either ACP or HYAFF 11 sponge. The osteochondral defect, 3 mm in diameter and 3 mm deep, was created in the weight-bearing region of the medial femoral condyle. The bone portion of the defect was first filled with ICP to a level approximately 1 mm below the articular surface. HA sponge (3 mm in diameter and 1-1.2 mm thick), with or without loading of autologous bone marrow-derived progenitor cells (MPCs), was then inserted into the defect on top of the ICP as it hardened. Animals were allowed free cage activity postoperatively, and killed 4 or 12 weeks (for the HYAFF 11 sponge group) after the surgery. At 4 weeks, histological examination showed that the defect was filled up to 90-100% of its depth. Whitish repair tissue on the top appeared to be integrated with the surrounding articular cartilage. Four distinct zones of repair tissue were identified: a superficial layer, a chondroid tissue layer, an interface between HA sponge and ICP, and the ICP material. Evidence of extensive osteoclastic and osteoblastic activities was observed in the bone tissue surrounding the defect edge and in ICP material. By 12 weeks, the zonal features of the repair tissue became more distinct; chondrocytes were arranged in a columnar array, and a calcified layer of cartilage was formed beneath the chondroid tissue in some specimens. The healing tissue of the HA sponge material loaded with MPCs had higher cellular density and better integration with the surrounding cartilage than HA sponge material not loaded with MPCs. This study suggests that using a two-phase composite graft may hold potential for the repair of osteochondral defects by providing mechanical support that mimicks subchondral bone, while also providing a chondrogenic scaffold for the top cartilage repair.

Treatment of osteochondral defects with autologous bone marrow in a hyaluronan-based delivery vehicle.

The natural repair of osteochondral defects can be enhanced with biocompatible, biodegradable and bioactive materials that provide structural support and molecular cuing to stimulate repair. Since bone marrow contains osteochondral progenitor cells and bioactive agents, it is hypothesized that the combination of scaffold and bone marrow would be a superior composite material for osteochondral repair. This hypothesis will be tested by comparing the outcome of osteochondral defects filled with a fibronectin-coated hyaluronan-based sponge (ACP) with or without autologous bone marrow. Thirty-three 4-month-old rabbits received 3-mm diameter osteochondral defects that were then filled with ACP loaded or not with autologous bone marrow. Rabbits were sacrificed at 2, 3, 4, 12, and 24 weeks after surgery and the condyles processed for histologic and immunohistochemical evaluation. The defects were graded with a histologic scoring scale. Except for the 3-week specimens, the histologic appearance of the defects was similar in both groups. Four weeks after surgery, the defects were filled with bone with a top layer of cartilage well integrated with the adjacent cartilage. Twelve and 24 weeks after surgery, the defects again showed bone filling. The primary difference between the 4-week samples and the 12- and 24-week samples was that the layer of cartilage that appeared to be thinner than the adjacent cartilage. At each harvest time, the overall histologic scores of the specimens did not reveal statistical differences between the treatment groups. However, as revealed by the results of the 3-week sacrifices, bone marrow loading appeared to accelerate the first stages of the repair process. The fibronectin-coated hyaluronan-based scaffold appears to organize the natural response and facilitate the integration of the neo-cartilage with the adjacent tissue. The fundamental tissue engineering principles derived from this study should provide guidelines for the development of comparable clinical reconstructive therapies.

Controlled release of hyaluronan oligomers from biodegradable polymeric microparticle carriers.

In the present study, biodegradable microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) were explored as a potential carrier for the controlled release of polysaccharide oligomers. To this end, hyaluronan (HY) oligomers of varying molecular weights were incorporated into PLGA/PEG microparticles. Using a two-level fractional factorial experimental design, four microparticle formulation parameters, the amount of PEG included in the microparticles, the initial HY loading of the microparticles, the molecular weight of HY, and the molecular weight of PLGA, were studied for their influence on the incorporation and in vitro release of HY over the period of 28 days. The entrapment efficiencies were found to range between 10+/-1% and 24+/-2% depending on the initial loading and the molecular weight of the HY oligomer used in the fabrication of the microparticles. The HY was released in a multiphasic fashion including an initial burst release, followed by two separate periods of linear release. The normalized cumulative mass released during the burst release ranged from 25.1+/-9.2% to 93.0+/-0.7% and was found to be significantly influenced by the initial HY loading, the HY molecular weight, and the PLGA molecular weight. The initial period of linear release lasted from day 1 to day 14 and displayed normalized cumulative rates of release from 0.1+/-0.0%/day to 1.4+/-0.2%/day. During this period, PEG content of the microparticles and HY molecular weight exerted the greatest influence on the rate of release. Finally, the second period of linear release lasted through the final time-point at day 28. Here, the normalized cumulative rate of release values ranged from 0.2+/-0.1%/day to 3.6+/-0.7%/day and were dependent on all formulation parameters studied. These results demonstrate the potential of PLGA/PEG blend microparticles for the controlled release of HY oligomers.

High-efficiency nonviral transfection of primary chondrocytes.

The introduction of foreign DNA into mammalian cells is an essential investigative tool in molecular biology. Nonviral approaches to transfection offer the advantage of relatively simple vector design, production, and purification and, for tissue engineering applications, avoid many of the potential risks associated with virus-mediated transfection methods. Unfortunately, primary cells, and in particular chondrocytes, are notoriously refractory to conventional transfection approaches, and optimized transfection efficiencies in these cells are extremely low (1-1.5%). In this chapter, we present three protocols that have proved useful in transfecting primary chondrocytes at high efficiency (~70%). The first uses radiofrequency electroporation, a transfection method that frequently works extremely well in cell types that are difficult to transfect. It should be noted that electroporation is not limited to DNA but that essentially any molecule can be introduced into the cell using this approach. In addition to the primary protocol, we present two additional reliable, albeit less efficient backup protocols, the first using exponential decay electroporation and the second FuGENE 6 transfection.

Generation of pluripotent stem cells and their differentiation to the chondrocytic phenotype.

It is well documented that adult cartilage has minimal self-repair ability. Current methods for treatment of cartilage injury focus on the relief of pain and inflammation and have met with limited long-term success. In the forefront of new therapeutic approaches, autologous chondrocyte transplantation is still only applied to a very small percentage of the patient population. Our laboratory has focused on cartilage repair using progenitor cells and studied their differentiation into cartilage. Adult mesenchymal stem cells are an attractive candidate as progenitor cells for cartilage repair because of their documented osteogenic and chondrogenic potential, ease of harvest, and ease of expansion in culture; furthermore, their use will obviate the need for harvesting precious healthy cartilage from a patient to obtain autologous chondrocytes for transplantation. However, the need to induce chondrogenic differentiation in the mesenchymal stem cells is superposed on other technical issues associated with cartilage repair; this adds a level of complexity over using mature chondrocytes. This chapter focuses on the methods involved in the isolation of human mesenchymal stem cells and their differentiation along the chondrogenic lineage. Although we have the technology to accomplish chondrogenic differentiation of stem cells, much is still to be learned regarding the regulatory mechanisms controlling the lineage transitions and maturation of the cartilaginous tissue.