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Biomedical subjects

Jeffrey M Karp

Publications and source records attributed to Jeffrey M Karp.

16 recordsLinked to original sources
PubMed2006

Co-culture of human embryonic stem cells with murine embryonic fibroblasts on microwell-patterned substrates.

Human embryonic stem (hES) cells are generally cultured as cell clusters on top of a feeder layer formed by mitotically inactivated murine embryonic fibroblasts (MEFs) to maintain their undifferentiated state. This co-culture system, which is typically used to expand the population of undifferentiated hES cells, presents several challenges since it is difficult to control cell cluster size. Large cell clusters tend to differentiate at the borders, and clusters with different sizes may lead to heterogeneous differentiation patterns within embryoid bodies. In this work, we develop a new approach to culture hES cells with controlled cluster size and number through merging microfabrication, and biomaterials technologies. Polymeric microwells were fabricated and used to control the size and uniformity of hES cell clusters in co-culture with MEFs. The results show that it is possible to culture hES cells homogeneously while keeping their undifferentiated state as confirmed by the expression of stem cell markers octamer binding protein 4 (Oct-4) and alkaline phosphatase (ALP). In addition, these clusters can be recovered from the microwells to generate nearly homogeneous cell aggregates for differentiation experiments.

Animals↗
PubMed2006

Micromolding of shape-controlled, harvestable cell-laden hydrogels.

Encapsulation of mammalian cells within hydrogels has great utility for a variety of applications ranging from tissue engineering to cell-based assays. In this work, we present a technique to encapsulate live cells in three-dimensional (3D) microscale hydrogels (microgels) of controlled shapes and sizes in the form of harvestable free standing units. Cells were suspended in methacrylated hyaluronic acid (MeHA) or poly(ethylene glycol) diacrylate (PEGDA) hydrogel precursor solution containing photoinitiator, micromolded using a hydrophilic poly(dimethylsiloxane) (PDMS) stamp, and crosslinked using ultraviolet (UV) radiation. By controlling the features on the PDMS stamp, the size and shape of the molded hydrogels were controlled. Cells within microgels were well distributed and remained viable. These shape-specific microgels could be easily retrieved, cultured and potentially assembled to generate structures with controlled spatial distribution of multiple cell types. Further development of this technique may lead to applications in 3D co-cultures for tissue/organ regeneration and cell-based assays in which it is important to mimic the architectural intricacies of physiological cell-cell interactions.

Animals↗
PubMed2006

A photolithographic method to create cellular micropatterns.

Here we describe a simple and rapid system for creation of patterned cell culture substrates. This technique is based on (1) printing a mask on a standard overhead transparency, (2) coating a thin layer of a photocrosslinkable chitosan on a slide, (3) exposing the slide and mask to ultraviolet (UV) light, and (4) rinsing the uncrosslinked polymer to expose the underlying cell-repellent patterns. Photocross-linkable chitosan does not require photoinitiators, it is non-toxic and forms flexible, biocompatible hydrogel upon short ( approximately min) UV exposure. Patterns of various shapes (lanes, squares, triangles, circles) were created on two surfaces commonly used for cell culture: glass and tissue culture polystyrene. The pattern size could be varied with a mum resolution using a single mask and varying UV exposure time. Cardiac fibroblasts formed stable patterns for up to 18 days in culture. Cardiomyocytes, patterned in lanes 68-99 microm wide, exhibited expression of cardiac Troponin I, well developed contractile apparatus and they contracted synchronously in response to electrical field stimulation. Osteoblasts (SAOS-2) localized in the exposed glass regions (squares, triangles, or circles; 0.063-0.5mm(2)). They proliferated to confluence in 5 days, expressed alkaline phosphatase and produced a mineralized matrix.

Animals↗
PubMed2006

Platelet releasate increases the proliferation and migration of bone marrow-derived cells cultured under osteogenic conditions.

Concentrated platelets and their products are currently being used as a clinical tool to accelerate endosseous wound healing. However, there is little understanding regarding the actions of platelets and platelet-released products on osteogenic cells. We show, herein, that releasate from thrombin-activated platelets increases the migration and proliferation of osteogenic cultures of bone marrow cells. Using a scratch wound assay, we demonstrated that platelet releasate (PR) stimulated up to a 2.4 +/- 0.5-fold increase in wound closure in serum-free medium, relative to a control containing thrombin. In the presence of serum, the addition of PR resulted in a 1.45 +/- 0.13-fold increase in scratch closure. To isolate cell migration from the effects of cell proliferation, cell monolayers were pre-incubated with 5, 10 and 20 microg/ml of Mitomycin C (MMC), which is a potent inhibitor of cell proliferation. This resulted in a large decrease in the leading front of scratch closure, which indicates that PR stimulated cell mitogenesis. However, irrespective of MMC pre-treatment, PR stimulated a motogenic response. These results provide evidence of possible mechanisms by which platelets could influence bone regeneration.

Alkylating Agents↗
PubMed2006

Interplay of biomaterials and micro-scale technologies for advancing biomedical applications.

Micro-scale technologies have already dramatically changed our society through their use in the microelectronics and telecommunications industries. Today these engineering tools are also useful for many biological applications ranging from drug delivery to DNA sequencing, since they can be used to fabricate small features at a low cost and in a reproducible manner. The discovery and development of new biomaterials aid in the advancement of these micro-scale technologies, which in turn contribute to the engineering and generation of new, custom-designed biomaterials with desired properties. This review aims to present an overview of the merger of micro-scale technologies and biomaterials in two-dimensional (2D) surface patterning, device fabrication and three-dimensional (3D) tissue-engineering applications.

Base Sequence↗
PubMed2006

Dental treatment of patients with long QT syndrome.

BACKGROUND: Long QT syndrome (LQTS) is associated with life-threatening cardiac arrhythmias causing syncope and sudden cardiac death, frequently precipitated by physical or psychological stress. TYPES OF STUDIES REVIEWED: The authors did a literature review of data published in peer-reviewed medical and dental journals. They also extracted epidemiologic information, correlations between genetic mutations and disease onset and progression, and data regarding outcomes of therapy from published peer-reviewed studies and the cohort population belonging to the International Long QT Syndrome Registry. RESULTS: LQTS is diagnosed after an unexpected cardiac event or through QT interval prolongation on an electrocardiogram. Gene mutation identification in LQTS provides insight into respective proarrhythmogenic factors and indicated therapeutic regimens. beta-blockers are the initial treatment for two of the three major forms of LQTS. Patients refractory to beta-antiadrenergic therapy may benefit from one or more of the following: cardiac pacemakers, implanted cardioverter defibrillators and left cardiac sympathetic denervation. CONCLUSIONS: Clinical studies are needed to investigate the safety of treating patients in an ambulatory setting. CLINICAL IMPLICATIONS: Preventive measures are recommended, including evaluation by a cardiac specialist before any dental intervention, use of anxiolytic protocols, avoidance of drugs that prolong the QT interval, and provision of treatment in a setting in which medical emergencies can be managed expeditiously. Dental treatment in a hospital and use of a general anesthetic administered by anesthesiologists are recommended for procedures in which anxiety and adrenergic stimulation would not be suppressed sufficiently in an ambulatory environment.

Adrenergic beta-Antagonists↗
PubMed2006

Nanoparticle-aptamer bioconjugates for cancer targeting.

The combination of targeted drug delivery and controlled-release technology may pave the road for more effective yet safer chemotherapeutic options for cancer therapy. Drug-encapsulated polymeric nanoparticle-aptamer bioconjugates represent an emerging technology that can facilitate the delivery of chemotherapeutics to primary and metastatic tumours. Aptamers are short nucleic acid molecules with binding properties and biochemical characteristics that may make them suitable for use as targeting molecules. The goal of this review is to summarise the key components that are required for creating effective cancer targeting nanoparticle-aptamer bioconjugates. The field of controlled release and the structure and properties of aptamers, as well as the criteria for constructing effective conjugates, will be discussed.

Animals↗
PubMed2005

Cultivation of human embryonic stem cells without the embryoid body step enhances osteogenesis in vitro.

Osteogenic cultures of embryonic stem cells (ESCs) are predominately derived from three-dimensional cell spheroids called embryoid bodies (EBs). An alternative method that has been attempted and merits further attention avoids EBs through the immediate separation of ESC colonies into single cells. However, this method has not been well characterized and the effect of omitting the EB step is unknown. Herein, we report that culturing human embryonic stem cells (hESCs) without the EB stage leads to a sevenfold greater number of osteogenic cells and to spontaneous bone nodule formation after 10-12 days. In contrast, when hESCs were differentiated as EBs for 5 days followed by plating of single cells, bone nodules formed after 4 weeks only in the presence of dexamethasone. Furthermore, regardless of the inclusion of EBs, bone matrix formed, including cement line matrix and mineralized collagen, which displayed apatitic mineral (PO4) with calcium-to-phosphorous ratios similar to those of hydroxyapatite and human bone. Together these results demonstrate that culturing hESCs without an EB step can be used to derive large quantities of functional osteogenic cells for bone tissue engineering.

Alkaline Phosphatase↗
PubMed2005

Sanguinarine activates polycyclic aromatic hydrocarbon associated metabolic pathways in human oral keratinocytes and tissues.

Sanguinarine's use in human clinical applications is currently controversial. While some studies have demonstrated sanguinarine's anti-inflammatory and anti-oxidant properties, other investigations reported sanguinarine's procarcinogenic effects. Like the tobacco-associated carcinogen, benzo(a)pyrene (B(a)P), sanguinarine is a polycyclic aromatic hydrocarbon (PAH). PAH exposure activates the aryl hydrocarbon transcription activating factor (AhR), resulting in nuclear translocation, binding to the aryl hydrocarbon nuclear translocator (ARNT), which thereby increases expression of a pool of carcinogen metabolizing enzymes. The goal of this study was to investigate whether sanguinarine activates this PAH-associated signaling cascade in human oral cells and tissues. Our results demonstrate that sanguinarine: (i) results in formation of the AhR-ARNT complex, (ii) induces AhR-associated gene expression, (iii) inhibits cytochrome P450 1A1 (CYP 1A1) microsomal oxidative activity and (iv) pretreatment upregulates CYP 1A1 function. Collectively, these data provide evidence that sanguinarine activates PAH-associated signaling and metabolic pathways. Notably, previous studies have demonstrated that mammalian hepatic microsomes metabolize sanguinarine to a mutagenic epoxide. Persons who respond to sanguinarine exposure with induction of primarily Phase I relative to Phase II enzymes are, therefore, at risk for sanguinarine bioactivation and its potential mutagenic effects.

Adult↗
PubMed2005

Thrombin mediated migration of osteogenic cells.

Given that thrombin is ubiquitously expressed at sites of vascular injury, and that osteogenic cells express receptors for thrombin, we questioned whether thrombin could attract osteogenic cells to a bony wound. Using a scratch wound assay, thrombin stimulated a significant increase in migration of osteogenic cultures of primary marrow cells. This effect was dependent on thrombin proteolytic activity; however, thrombin was unable to stimulate the migration of a more differentiated marrow-derived osteogenic cell line. To better understand the role of thrombin in osteoprogenitor migration, we developed an osteoprogenitor migration assay that combines a modified Boyden chamber with a bone nodule assay. Primary cells that migrated through the transwell filter in the presence of thrombin formed significantly more bone nodules compared to the condition without thrombin. This was not due to proliferation or differentiation effects of thrombin. In contrast, thrombin was unable to stimulate an increase in the number of nodules for the more differentiated osteogenic cell line. Thus, our results suggest that thrombin exhibits differential motogenic effects on osteogenic cells depending on their differentiation state. The cell migration/bone nodule assay described here is the first assay that can be specifically used to examine the effects of factors on the migration of osteoprogenitor cells, particularly those derived from primary populations.

Actins↗
PubMed2004

Fibrin-filled scaffolds for bone-tissue engineering: An in vivo study.

Recently, fibrin sealants that typically contain supra physiological concentrations of fibrinogen and thrombin have been investigated as matrices to facilitate the delivery of cells within biodegradable scaffolds for tissue engineering applications. It is well known from in vitro experiments that the thrombin concentration present during fibrin polymerization influences the structural properties of fibrin, and these can affect cell invasion. This study was conducted to determine whether the structural properties of fibrin can affect bony wound healing in vivo. Drill hole defects were created in the distal femurs of 20 rats. Four experimental groups were used: nontreated defects, scaffolds alone, and scaffolds filled with fibrin polymerized with either a low thrombin concentration [fibrin(low T)] or a high thrombin concentration [fibrin(high T)]. The area of bone formed at 2, 5, and 11 days after implantation was determined histomorphometrically. After 5 days, scaffolds filled with fibrin(high T) were infiltrated with less bone than empty scaffolds (p < 0.05), but no statistical difference was found between the empty scaffolds and the scaffolds filled with fibrin(low T). After 11 days, both fibrin-filled scaffolds significantly delayed bony wound healing (p < 0.004). Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the two fibrin formulations showed no difference in gamma-gamma crosslink formation. This work demonstrates that fibrin sealants in their present state are not ideal for enhancing bone-tissue invasion into scaffolds, and that the structural properties of fibrin matrices may be an important design parameter for maximizing host tissue invasion during wound healing.

Animals↗
PubMed2004

Opinions and trends in biomaterials education: report of a 2003 Society for Biomaterials survey.

The Society for Biomaterials (SFB) aims to serve its members through acting as a forum for the exchange of information and ideas. To aid in the practical development of the SFB and more specifically biomaterials education, all active, associate, and student members were surveyed. In general, the survey asked questions regarding respondent demographics, experiences and activities with the SFB, and opinions about biomaterials education. Perceptions and needs of biomaterials-related education and career-related training practices were a specific focus of the survey. A total of 140 individuals responded to the survey for a response rate of 18%. Members from industry felt that new hires, in general, should be better trained in product development, regulatory issues for new materials and devices, and in the relevant testing required. When asked what was missing from their professional education, many respondents commented that business training in areas such as negotiations, management, and understanding the needs outside of academia was lacking. Also, many respondents seemed to have trouble identifying with what they were supposed to know and felt a "lack of set professional knowledge." This study has raised many ideas and questions that require further discussion. The results should ultimately be useful for helping the SFB decide how best to focus future efforts in biomaterials education.

Biocompatible Materials↗
PubMed2003

Bone formation on two-dimensional poly(DL-lactide-co-glycolide) (PLGA) films and three-dimensional PLGA tissue engineering scaffolds in vitro.

For some bone tissue engineering strategies, direct contact of newly synthesized bone with a scaffold is important for structural continuity and stability at the scaffold/bone interface. Thus, as the polymer degrades, the support function of the scaffold could be adopted by the developing bone structure. This study was designed to determine whether poly(DL-lactide-co-glycolide) with a comonomer ratio of 75:25 supports bone apposition in vitro. Osteogenic cells derived from rat bone marrow cells were cultured for 2 weeks on polymeric two-dimensional films and three-dimensional tissue engineering scaffolds. Bacteriological grade polystyrene and tissue culture polystyrene dishes served as negative and positive controls for interfacial bone deposition, respectively. The surfaces of the prepared substrates were characterized by X-ray photoelectron spectroscopy, dynamic water contact angle, scanning electron microscopy, and atomic force microscopy. After cell culture, the elaborated matrix was examined using scanning electron microscopy, transmission electron microscopy, and energy dispersive spectroscopy. The results show that poly(DL-lactide-co-glycolide) supports appositional bone growth on both two-dimensional films and three-dimensional scaffolds, including the formation of a mineralized cement line matrix. Furthermore, surface topographical features are not required for the adherence of the cement line matrix to the polymer.

Animals↗
PubMed2003

Effect of platelet releasate on bone cell migration and recruitment in vitro.

The use of platelets and platelet products has become increasingly popular clinically as a means of accelerating endosseous wound healing. It is likely that growth factors released by activated platelets at the site of injury play a role in bone regeneration by stimulating the migration and proliferation of bone cells. In this study, a novel in vitro assay was developed to study the effects of platelet releasate (PR) collected from activated platelet concentrate on rat bone marrow-derived cells. Cultures of primary rat bone marrow cells were overlaid with a fibrin matrix, and the number of cells migrating within the three-dimensional matrix and the leading front of migration were quantified. The addition of PR to the top of the fibrin gels at different time points caused a 25% increase in the leading front of migration and a 3.5-fold increase in the number of migrating cells. Platelet releasate was also shown to have a mitogenic effect on bone cells in proliferation studies. Comparison between migration and proliferation data indicated that PR stimulates the initial recruitment of bone marrow cells to migration. This assay further allowed the determination that rat bone marrow cells are capable of exerting contractile forces on fibrin matrices and that matrix contraction is directly related to the migratory activity of cells. The results provide a potential mechanism to explain why biologically active platelet-derived factors enhance endosseous wound healing.

Animals↗
PubMed2003

Fabrication of precise cylindrical three-dimensional tissue engineering scaffolds for in vitro and in vivo bone engineering applications.

It is sometimes necessary to form highly porous polymeric tissue engineering scaffolds into various shapes and sizes. Ideally, in these cases, the three-dimensional morphology should be maintained to the outer margins of the scaffold so as to provide optimum function. Many biodegradable polymeric scaffolds are soft and delicate, however, and their poor physical strength presents a challenge when cutting these materials into the required shapes. We describe a simple device that can be used quickly and accurately to cut cylindrical shapes from such delicate polymeric scaffold materials, which maintain their morphological features to the margins of the shapes produced. We demonstrate that the device can be used to create scaffolds with reproducible dimensions having an SD in mass of less then 6%. The in vitro utility of scaffolds cut with the device was established through demonstrating bone marrow-derived cell invasion into fibrin-filled scaffolds that fit precisely into the wells of 24-well plates. We also demonstrate the in vivo utility of precise cylindrically shaped scaffolds by observing rapid bone invasion into 2.4-mm diameter scaffolds that have been placed into drill hole defects in the distal femur of young rats. When scaffolds are filled with fibrin before implantation as part of a bone tissue engineering strategy, less blood fills the defect site and the fibrin is gradually remodeled and replaced by bone. The ability to cut precise cylindrical scaffolds in the millimeter size range has allowed for the creation of a new small animal model that may prove useful for screening tissue engineering scaffolds for further study.

Animals↗