Biomedical subjects

Eben Alsberg

Publications and source records attributed to Eben Alsberg.

11 recordsLinked to original sources

PubMed2005

Novel dynamic rheological behavior of individual focal adhesions measured within single cells using electromagnetic pulling cytometry.

The rheology of cells and sub-cellular structures, such as focal adhesions, are important for cell form and function. Here we describe electromagnetic pulling cytometry (EPC), a technique to analyze cell rheology by applying dynamic tensional forces to ligand-coated magnetic microbeads bound to cell surface integrin receptors. EPC utilizes an electromagnetic microneedle that is integrated with a computerized control and image acquisition system and an inverted microscope and CCD camera to monitor bead displacement. Arbitrary force regimens may be defined over a wide range of frequency (DC to 10 Hz) and force (100 pN to 10 nN). With EPC, the viscoelastic creep response of individual focal adhesions was measured over three decades in time using RGD-coated magnetic microbeads bound to integrins that induce local focal adhesion assembly and coupling to the internal cytoskeleton. These data were compared to the power-law-like predictions from the soft glassy model of cell rheology proposed by Fabry et al. Although power-law-like behavior was observed in some focal adhesions, 52% of these structures did not exhibit power-law-like behavior, but instead exhibited either a multi-phase response characterized by abrupt changes in slope or experienced a retraction in the opposite direction to the applied force, especially in response to prolonged force application. These data suggest that while the soft glassy model may provide reasonable estimates for aggregate mechanical behavior of living cells, the rheological behavior of individual focal adhesions may be more heterogeneous and complex than suggested by the soft glassy model. These results are considered in context with the hierarchical nature of cytoskeletal architecture.

Animals↗

PubMed2005

FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness.

The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

3T3 Cells↗

PubMed2005

Control of basement membrane remodeling and epithelial branching morphogenesis in embryonic lung by Rho and cytoskeletal tension.

Local alterations in the mechanical compliance of the basement membrane that alter the level of isometric tension in the cell have been postulated to influence tissue morphogenesis. To explore whether cell tension contributes to tissue pattern formation in vivo, we modulated cytoskeletal force generation in embryonic mouse lung (embryonic days 12-14) rudiments using inhibitors of Rho-associated kinase (ROCK), myosin light chain kinase, myosin ATPase, and microfilament integrity, or a Rho stimulator (cytotoxic necrotizing factor-1). Tension inhibition resulted in loss of normal differentials in basement membrane thickness, inhibition of new terminal bud formation, and disorganization of epithelial growth patterns as well as disruption of capillary blood vessels. In contrast, increasing cell tension through Rho activation, as confirmed by quantitation of myosin light chain phosphorylation and immunohistocytochemical analysis of actin organization, accelerated lung branching and increase capillary elongation. These data suggest that changes in cytoskeletal tension mediated by Rho signaling through ROCK may play an important role in the establishment of the spatial differentials in cell growth and extracellular matrix remodeling that drive embryonic lung development.

Actins↗

PubMed2004

Shape-defining scaffolds for minimally invasive tissue engineering.

BACKGROUND: Minimally invasive surgical procedures are increasingly important in medicine, but biomaterials consistent with this delivery approach that allow one to control the structure of the material after implantation are lacking. Biomaterials with shape-memorizing properties could permit minimally invasive delivery of cell transplantation constructs and enable the formation of new tissues or structures in vivo in desired shapes and sizes. METHODS: Macroporous alginate hydrogel scaffolds were prepared in a number of predefined geometries, compressed into significantly smaller, different "temporary" forms, and introduced into immunocompromised mice by means of minimally invasive surgical delivery through a small catheter. Scaffolds were rehydrated in situ with a suspension of cells (primary bovine articular chondrocytes) or cell-free medium and delivered through the same catheter. Specimens were harvested at 1 hr to evaluate the efficacy of cell delivery and the recovery of scaffold geometry, and at 8 and 24 weeks to evaluate neotissue formation. RESULTS: A high percentage (88%) of scaffolds that were introduced with a catheter and rehydrated with cells had recovered their original shape and size within 1 hr. This delivery procedure resulted in cartilage structures with the geometry of the original scaffold by 2 months and histologically mature appearing tissue at 6 months. CONCLUSIONS: Shaped hydrogels, formed by covalently cross-linking, can be structurally collapsed into smaller, temporary shapes that permit their minimally invasive delivery in vivo. The rapid recovery of scaffold properties facilitates efficient cell seeding in vivo and permits neotissue formation in desired geometries.

Alginates↗

PubMed2004

Tracheal reconstruction using tissue-engineered cartilage.

OBJECTIVES: To determine whether rabbit cartilage can be tissue engineered using a polyglycolic acid (PGA) construct composed of PGA mesh, autologous chondrocytes, and alginate covalently linked with the cell adhesion sequence arginine-glycine-aspartic acid (RGD), and to investigate the feasibility of reconstructing tracheal defects using the PGA construct in conjunction with a bioabsorbable intratracheal stent. METHODS: Nineteen New Zealand White rabbits were used. Nine rabbits underwent subcutaneous implantation of 3 different PGA construct combinations: (1) PGA, autologous chondrocytes, and RGD-modified alginate; (2) PGA, autologous chondrocytes, and unmodified alginate; and (3) PGA and RGD-modified alginate. The remaining 10 animals underwent anterior tracheal reconstruction using fascia lata grafts and the complete PGA construct (PGA, autologous chondrocytes, and RGD-modified alginate). At the time of tracheal reconstruction, a poly-l-lactic acid intratracheal stent was placed in 5 of these latter animals. Rates of tracheal stenosis and mortality were compared with those of historical control animals. Histologic analysis was performed on the PGA constructs. RESULTS: In the subcutaneous implants, the PGA constructs made with chondrocytes (with and without RGD) demonstrated mature cartilage formation in 7 (78%) of the 9 animals. No cartilage was seen in PGA constructs made without chondrocytes. Two of the 10 animals that underwent tracheal reconstruction with the complete PGA construct survived to 20 weeks and demonstrated patent airways, 1 with a stent and 1 without a stent (80% overall mortality). Histologic analysis showed mature cartilage formation at the tracheal reconstruction site. Historical control animals that underwent reconstruction with fascia lata alone demonstrated the lowest overall mortality. CONCLUSIONS: Cartilage can be tissue engineered in rabbits using PGA mesh embedded with alginate-encapsulated autologous chondrocytes. It is also possible to reconstruct tracheal defects with this method of cartilage engineering, although the mortality rate in this study is high.

Alginates↗

PubMed2004

Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells.

Supraphysiological concentrations of exogenous growth factors are typically required to obtain bone regeneration, and it is unclear why lower levels are not effective. We hypothesized that delivery of bone progenitor cells along with appropriate combinations of growth factors and scaffold characteristics would allow physiological doses of proteins to be used for therapeutic bone regeneration. We tested this hypothesis by measuring bone formation by rat bone marrow stromal cells (BMSCs) transplanted ectopically in SCID mice using alginate hydrogels. The alginate was gamma-irradiated to vary the degradation rate and then covalently modified with RGD-containing peptides to control cell behavior. In the same delivery vehicle, we incorporated bone morphogenetic protein-2 (BMP2) and transforming growth factor-beta3 (TGF-beta3), either individually or in combination. Individual delivery of BMP2 or TGF-beta3 resulted in negligible bone tissue formation up to 22 weeks, regardless of the implant degradation rate. In contrast, when growth factors were delivered together from readily degradable hydrogels, there was significant bone formation by the transplanted BMSCs as early as 6 weeks after implantation. Furthermore, bone formation, which appeared to occur by endochondral ossification, was achieved with the dual growth factor condition at protein concentrations that were more than an order of magnitude less than those reported previously to be necessary for bone formation. These data demonstrate that appropriate combinations of soluble and biomaterial-mediated regulatory signals in cell-based tissue engineering systems can result in both more efficient and more effective tissue regeneration.

Animals↗

PubMed2004

Shape retaining injectable hydrogels for minimally invasive bulking.

PURPOSE: Particle migration, poor shape definition and/or rapid resorption limit the success of current urethral bulking agents. We propose that shape defining porous scaffolds that allow cell infiltration and anchoring, and may be delivered in a minimally invasive manner may provide many advantageous features. MATERIALS AND METHODS: Alginate hydrogels were prepared with varying degrees of covalent cross-linking and different pore characteristics. Dehydrated scaffolds were compressed into smaller, temporary forms, introduced into the dorsal subcutaneous space of CD-1 mice by minimally invasive delivery through a 10 gauge angiocatheter and rehydrated in situ with a saline solution delivered through the same catheter. Ionically cross-linked calcium alginate gel served as a control. Specimens were harvested at 2, 6, 12 and 24 weeks to evaluate implant shape retention and volume, cell infiltration and calcification, and the presence of an inflammatory response. RESULTS: A total of 90 scaffolds were implanted and 95% were recovered at the site of injection. All of these scaffolds successfully rehydrated and 80% recovered and maintained their original 3-dimensional shape for 6 months. Scaffold volume and tissue infiltration varied depending on the degree of alginate cross-linking. Highly cross-linked materials (20% and 35%) demonstrated the best volume maintenance with the latter facilitating the most tissue infiltration. The inflammatory response was minimal except with the 80% cross-linked material. Calcification was not observed in covalently cross-linked scaffolds. In contrast, 98% of calcium alginate implants were calcified. CONCLUSIONS: Shape retaining porous hydrogels meet many of the requirements necessary for a successful injectable bulking agent and offer advantages over currently used agents.

Alginates↗

PubMed2004

Tissue-engineered small intestine improves recovery after massive small bowel resection.

OBJECTIVE: Rescue with tissue-engineered small intestine (TESI) after massive small bowel resection (MSBR). SUMMARY BACKGROUND DATA: Short bowel syndrome is a morbid product of massive small bowel resection. We report the first replacement of a vital organ by tissue engineering with TESI after MSBR. METHODS: Ten male Lewis rats underwent TESI implantation with green fluorescent protein (GFP)-marked cells (TESI+, n = 5) or sham laparotomy (TESI-, n = 5) followed by MSBR. Side-to-side anastomosis of TESI to proximal small intestine was performed or omitted. TESIO animals underwent implantation of engineered intestine with no further surgery. Weights were measured QOD until day 40. Transit times were measured. DNA assay was performed with computer morphometry. Northern blots of RNA were probed for intestinal alkaline phosphatase (IAP) and villin. Hematoxylin and eosin, S100, and smooth muscle actin immunohistochemistry were performed. Blood was collected at sacrifice. RESULTS: All 10 rats initially lost then regained weight. The initial rate of weight loss was higher in TESI+ versus TESI-, but the nadir was reached a week earlier with more rapid weight gain subsequently to 98% preoperative weight on day 40 in animals with engineered intestine versus 76% (P < 0.03). Serum B12 was higher at 439 pg/mL versus 195.4 pg/mL. IAP mRNA appeared greater in TESI+ than TESIO, with constant villin levels. Histology revealed appropriate architecture including nerve. GFP labeling persisted. CONCLUSIONS: Anastomosis of TESI significantly improved postoperative weight and B12 absorption after MSBR. IAP, a marker of differentiation in intestinal epithelium, is present in TESI, and GFP labeling was accomplished.

Actins↗

PubMed2003

Tissue-engineered large intestine resembles native colon with appropriate in vitro physiology and architecture.

OBJECTIVE: Novel production and in vitro characterization of tissue engineered colon. SUMMARY BACKGROUND DATA: The colon provides important functions of short chain fatty acid production, sodium and water absorption, and storage. We report the first instance of tissue-engineered colon (TEC) production from autologous cells and its in vitro characterization. METHODS: Organoid units, mesenchymal cell cores surrounded by a polarized epithelia derived from full thickness sigmoid colon dissection from neonatal Lewis rats, adult rats, and tissue engineered colon itself, were implanted on a polymer scaffold into the omentum of syngeneic hosts. TEC was either anastomosed at 4 weeks or excised for Ussing chamber studies or histology, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling assay. RESULTS: TEC was generated by 100% of all animals without regard to tissue source, the first instance of engineered intestine from adult cells or an engineered tissue. TEC architecture is identical to native with muscularis propria staining for actin, acetylcholinesterase detected in a linear distribution in the lamina propria, S100-positive cells, ganglion cells, and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling assay similar to native colon. Ussing chamber data indicated in vitro function consistent with mature colonocytes, and a positive short circuit current response to theophylline indicating intact ion transfer. TEM showed normal microarchitecture. Colon architecture was maintained in anastomosis with gross visualization of fluid uptake. CONCLUSIONS: TEC can be successfully produced with fidelity to native architecture and in vitro function from neonatal syngeneic tissue, adult tissue, and TEC itself.

Animals↗

PubMed2002

Engineering growing tissues.

Regenerating or engineering new tissues and organs may one day allow routine replacement of lost or failing tissues and organs. However, these engineered tissues must not only grow to fill a defect and integrate with the host tissue, but often they must also grow in concert with the changing needs of the body over time. We hypothesized that tissues capable of growing with time could be engineered by supplying growth stimulus signals to cells from the biomaterial used for cell transplantation. In this study, chondrocytes and osteoblasts were cotransplanted on hydrogels modified with an RGD-containing peptide sequence to promote cell multiplication. New bone tissue was formed that grew in mass and cellularity by endochondral ossification in a manner similar to normal long-bone growth. Transplanted cells organized into structures that morphologically and functionally resembled growth plates. These engineered tissues could find utility in treating diseases and injuries of the growth plate, testing the effect of experimental drugs on growth-plate function and development, and investigating the biology of long-bone growth. Furthermore, this concept of promoting the growth of engineered tissues could find great utility in engineering numerous tissue types by way of the transplantation of a small number of precursor cells.

Alginates↗

PubMed2002

Tissue-engineered colon exhibits function in vivo.

BACKGROUND: Postcolectomy morbidities include important changes in enterohepatic circulation, stool microbiology, and absorption. The surgical substitution of an ileal pouch for the absent colon also has a number of serious complications. We report in vivo colon replacement by tissue-engineered colon (TEC) in lieu of an ileal pouch. METHODS: End-ileostomies were created in 22 male Lewis rats. In 11 animals, side-to-side ileum-TEC anastomosis was performed 1 cm from the stoma. This group was compared with end-ileostomy alone. Serial weights were measured, and animals were harvested sequentially for assessment of histologic signs of pouchitis. Transit times, stool dry and wet weights, and serum and stool colon function markers were collected. RESULTS: Animals survived 41 days. Weight loss was more than 1.5 times greater in the end-ileostomy alone group compared with the ileum-TEC group. Transit times were significantly longer in the ileum-TEC group than the end-ileostomy alone group, with lower stool moisture content and higher total serum bile acids. Animals without TEC had statistically significant hyponatremia, elevated serum urea nitrogen, and lower stool short chain fatty acids (13.5 micromol/kg vs 84.2) with an abnormal distribution. CONCLUSIONS: TEC successfully recapitulates some major physiologic functions of native large intestine in vivo.

Anastomosis, Surgical↗