Search

Mechanical stiffness and degradability are important material parameters in tissue engineering. The aim of this study was to address the hypothesis that these variables regulate the function of myoblasts cultured in 2-D and 3-D microenvironments. Development of cell-interactive alginate gels with tunable degradation rates and mechanical stiffness was established by a combination of partial oxidation and bimodal molecular weight distribution. Higher gel mechanical properties (13 to 45 kPa) increased myoblast adhesion, proliferation, and differentiation in a 2-D cell culture model. Primary mouse myoblasts were more highly responsive to this cue than the C2C12 myoblast cell line. Myoblasts were then encapsulated in gels varying in degradation rate to simultaneously investigate the effect of degradation and subsequent reduction of mechanical properties on cells in a 3-D environment. C2C12 cells in more rapidly degrading gels exhibited lower proliferation, as they exited the cell cycle to differentiate, compared to those in nondegradable gels. In contrast, mouse primary myoblasts illustrated significantly higher proliferation in degradable gels than in nondegradable gels, and exhibited minimal differentiation in either type of gel. Altogether, these studies suggest that a critical balance between material degradation rate and mechanical properties may be required to regulate formation of engineered skeletal muscle tissue, and that results obtained with the C2C12 cell line may not be predictive of the response of primary myoblasts to environmental cues. The principles delineated in these studies may be useful to tailor smart biomaterials that can be applied to many other polymeric systems and tissue types.

Blood vessels of the vertebrate circulatory system typically exhibit tissue-specific patterning. However, the cues that guide the development of these patterns remain unclear. We investigated the effect of cyclic uniaxial strain on vascular endothelial cell dynamics and sprout formation in vitro in two-dimensional (2D) and three-dimensional (3D) culture systems under the influence of growth factors. Cells preferentially aligned and moved in the direction perpendicular to the major strain axis in monolayer culture, and mechanical strain also regulated the spatial location of cell proliferation in 2D cell culture. Cells in 3D cell culture could be induced to form sprouts by exposure to appropriate growth factor combinations (vascular endothelial growth factor and hepatocyte growth factor), and the strain direction regulated the directionality of this process. Moreover, cyclic uniaxial strain inhibited branching of the structures formed by endothelial cells and increased their thickness. Taken together, these data support the importance of external mechanical stimulation in the regulation of endothelial cell migration, proliferation, and differentiation into primitive vessels.

The close apposition of osteoblasts and chondrocytes in bone and their interaction during bone development and regeneration suggest that they may each regulate the other's growth and differentiation. In these studies, osteoblasts and chondrocytes were co-cultured in vitro, with both direct and indirect contact. Proliferation of the co-cultured chondrocytes was enhanced using soluble factors produced from the osteoblasts, and the differentiation level of the osteoblasts influenced the differentiation level of the chondrocytes. In addition, the chondrocytes regulated differentiation of the co-cultured osteoblasts using soluble factors and direct contact. These data support the possibility of direct, reciprocal instructive interactions between chondrocytes and osteoblasts in a variety of normal processes and further suggest that it may be necessary to account for this signaling in the regeneration of complex tissues comprising cartilage and mineralized tissue.

Myoblast transplantation is currently limited by poor survival and integration of these cells into host musculature. Transplantation systems that enhance the viability of the cells and induce their outward migration to populate injured muscle may enhance the success of this approach to muscle regeneration. In this study, enriched populations of primary myoblasts were seeded onto delivery vehicles formed from alginate, and the role of vehicle design and local growth factor delivery in cell survival and migration were examined. Only 5 +/- 2.5% of cells seeded into nanoporous alginate gels survived for 24 h and only 4 +/- 0.5% migrated out of the gels. Coupling cell adhesion peptides (G4RGDSP) to the alginate prior to gelling slightly increased the viability of cells within the scaffold to 16 +/- 1.4% and outward migration to 6 +/- 1%. However, processing peptide-modified alginate gels to yield macroporous scaffolds, in combination with sustained delivery of HGF and FGF2 from the material, dramatically increased the viability of seeded cells over a 5-day time course and increased outward migration to 110 +/- 12%. This data indicate long-term survival and migration of myoblasts placed within polymeric delivery vehicles can be greatly increased by appropriate scaffold composition, architecture, and growth factor delivery. This system may be particularly useful in the regeneration of muscle tissue and be broadly useful in the regeneration of other tissues as well.

Tissue engineering approaches have been investigated as a strategy for hepatocyte transplantation; however the death of a majority of transplanted cells critically limits success of these approaches. In a previous study, a transient increase in hepatocyte survival was achieved through delivery of vascular endothelial growth factor (VEGF) from the porous polymer scaffold utilized for cell delivery. To enhance longer-term survival of the hepatocytes, this delivery system was modified to additionally deliver epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in a sustained manner. Hepatocytes were subcutaneously implanted in SCID mice on scaffolds containing EGF and/or HGF, in addition to VEGF, and survival was monitored for two weeks. A short-term enhancement of hepatocyte survival was observed after one week and is attributed to VEGF-enhanced vascularization, which was not altered by EGF or HGF. Surprisingly, long-term hepatocyte engraftment was not improved, as survival declined to the level of control conditions for all growth factor combinations after two weeks. This investigation indicates that the survival of hepatocytes transplanted into heterotopic locations is dependent on multiple signals. The delivery system developed for the current study may be useful in elucidating the specific factors controlling this process, and bring therapeutic transplantation of hepatocytes closer to implementation.

A developing therapy for complete or partial loss of function in various tissues and organs involves transplanting an appropriate cell population, capable of compensating for the existing deficiencies. Clinical application of this type of strategy is currently limited by the death or dedifferentiation of the transplanted cells after delivery to the recipient. A delay in thorough vascularization of the implant area creates an environment low in oxygen and other nutrients, and likely contributes to the initial death of transplanted cells. We have addressed this problem by sustained delivery of vascular endothelial growth factor (VEGF), an initiator of angiogenesis, from a porous polymer matrix utilized simultaneously for cell delivery. As expected from previous studies, VEGF delivered from these constructs elicited an enhanced angiogenic response over a 2-week period when implanted subcutaneously in SCID mice. Hepatocytes implanted using VEGF-containing matrices demonstrated significantly greater survival after 1 week in vivo as compared with cells implanted on matrices without growth factor. The results of this study therefore indicate that enhancing vascularization in the location of transplanted cells promotes their survival. In addition, this delivery system may be used in future studies to directly promote cell survival and function by also providing growth factors specific to the transplanted cells.

Maintenance of liver-specific function of hepatocytes in culture is still difficult. Improved culture conditions may enhance the cell growth and function of cultured cells. We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures. Hepatocytes were harvested from Lewis rats. Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system. Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix. Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period. Culture under flow condition significantly enhanced cell numbers. An additional improvement of this effect was observed, when matrix coating was used. Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture. Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function. Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect. Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.

One of the fundamental principles that underlies tissue-engineering strategies using cell transplantation is that a newly formed tissue must acquire and maintain sufficient vascularization in order to support its growth. Enhancing angiogenesis through delivery of growth factors is one approach to establishing a vascular network to these tissues. In this study, we tested the potential of bone marrow stromal cells (BMSCs) to modulate the growth and differentiation activities of blood vessel precursors, endothelial cells (ECs), by their secretion of soluble angiogenic factors. The growth and differentiation of cultured ECs were enhanced in response to exposure to BMSC conditioned medium (CM). Enzyme-linked immunosorbent assays demonstrated that both mouse and human BMSCs secreted significant quantities of vascular endothelial growth factor (VEGF) (2.4-3.1 ng/10(6) cells per day). Furthermore, eliminating the activity of BMSC-secreted VEGF with blocking antibodies completely blocked the CM effects on cultured ECs. These data demonstrate that human BMSCs secrete sufficient quantities of VEGF to enhance survival and differentiation of endothelial cells in vitro, and suggest they may be capable of directly orchestrating angiogenesis in vivo.

Radiation therapy for cancer in the head and neck region leads to a marked loss of salivary gland parenchyma, resulting in a severe reduction of salivary secretions. Currently, there is no satisfactory treatment for these patients. To address this problem, we are using both tissue engineering and gene transfer principles to develop an orally implantable, artificial fluid-secreting device. In the present study, we examined the tissue compatibility of two biodegradable substrata potentially useful in fabricating such a device. We implanted in Balb/c mice tubular scaffolds of poly-L-lactic acid (PLLA), poly-glycolic acid coated with PLLA (PGA/PLLA), or nothing (sham-operated controls) either beneath the skin on the back, a site widely used in earlier toxicity and biocompatibility studies, or adjacent to the buccal mucosa, a site quite different functionally and immunologically. At 1, 3, 7, 14, and 28 days postimplantation, implant sites were examined histologically, and systemic responses were assessed by conventional clinical chemistry and hematology analyses. Inflammatory responses in the connective tissue were similar regardless of site or type of polymer implant used. However, inflammatory reactions were shorter and without epithelioid and giant cells in sham-operated controls. Also, biodegradation proceeded more slowly with the PLLA tubules than with the PGA/PLLA tubules. No significant changes in clinical chemistry and hematology were seen due to the implantation of tubular scaffolds. These results indicate that the tissue responses to PLLA and PGA/PLLA scaffolds are generally similar in areas subjacent to skin in the back and oral cavity. However, these studies also identified several potentially significant concerns that must be addressed prior to initiating any clinical applications of this device.

Macroporous scaffolds composed of biodegradable polymers have found extensive use as three-dimensional substrates either for in vitro cell seeding followed by transplantation, or as conductive substrates for direct implantation in vivo. Methods abound for creation of macroporous scaffolds for tissue engineering, and common methods typically employ a solid porogen within a three-dimensional polymer matrix to create a well-defined pore size, pore structure, and total scaffold porosity. This study describes an approach to impart improved pore interconnectivity to polymer scaffolds for tissue engineering by partially fusing the solid porogen together prior to creation of a continuous polymer matrix. Three dimensional, porous scaffolds of the copolymer 85:15 poly(lactide-co-glycolide) were fabricated via either a solvent casting/particulate leaching process, or a gas foaming/particulate leaching process. Prior to creation of a continuous polymer matrix the NaCl crystals, which serve as the solid porogen, are partially fused via treatment in 95% humidity. Scanning electron micrographs clearly display fused salt crystals and an enhancement in pore interconnectivity in the salt fused scaffolds prepared via both solvent casting and gas foaming, and the extent of pore interconnectivity is enhanced with longer treatment times. Fusion of salt crystal for 24 h increased the radius of curvature of salt crystals, and led to a twofold increase in the compressive modulus of solvent cast scaffolds (total porosity of 97 +/- 1%). Fusion of NaCl crystals prior to gas foaming resulted in a decrease in scaffold compressive modulus from 277 +/- 60k Pa to 187 +/- 30k Pa (total porosity of 94 +/- 1%). The resulting highly interconnected scaffolds have implications for facilitated cell migration, abundant cell-cell interaction, and potentially improved neural and vascular growth within tissue engineering scaffolds.

Extreme salivary hypofunction is a result of tissue damage caused by irradiation therapy for cancer in the head and neck region. Unfortunately, there is no currently satisfactory treatment for this condition that affects up to 40,000 people in the United States every year. As a novel approach to managing this problem, we are attempting to develop an orally implantable, fluid-secreting device (an artificial salivary gland). We are using the well-studied HSG salivary cell line as a potential allogeneic graft cell for this device. One drawback of using a cell line is the potential for malignant transformation. If such an untoward response occurred, the device could be removed. However, in the event that any HSG cells escaped, we wished to provide additional patient protection. Accordingly, we have engineered HSG cells with a hybrid adeno-retroviral vector, AdLTR.CMV-tk, to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene as a novel safety factor. Cells were grown on plastic plates or on poly-L-lactic acid disks and then transduced with different multiplicities of infection (MOIs) of the hybrid vector. Thereafter, various concentrations of ganciclovir (GCV) were added, and cell viability was tested. Transduced HSG cells expressed HSV-tk and were sensitive to GCV treatment. Maximal effects were seen at a MOI of 10 with 50 microM of GCV, achieving 95% cell killing on the poly-L-lactic acid substrate. These results suggest that engineering the expression of a suicide gene in an allogeneic graft cell may provide additional safety for use in an artificial salivary gland device.

Bone regeneration is based on the hypothesis that healthy progenitor cells, either recruited or delivered to an injured site, can ultimately regenerate lost or damaged tissue. Three-dimensional porous polymer scaffolds may enhance bone regeneration by creating and maintaining a space that facilitates progenitor cell migration, proliferation, and differentiation. As an initial step to test this possibility, osteogenic cells were cultured on scaffolds fabricated from biodegradable polymers, and bone development on these scaffolds was evaluated. Porous polymer scaffolds were fabricated from biodegradable polymers of lactide and glycolide. MC3T3-E1 cells were statically seeded onto the polymer scaffolds and cultured in vitro in the presence of ascorbic acid and beta-glycerol phosphate. The cells proliferated during the first 4 weeks in culture and formed a space-filling tissue. Collagen messenger RNA levels remained high in these cells throughout the time in culture, which is consistent with an observed increase in collagen deposition on the polymer scaffold. Mineralization of the deposited collagen was initially observed at 4 weeks and subsequently increased. The onset of mineralization corresponded to increased mRNA levels for two osteoblast-specific genes: osteocalcin and bone sialoprotein. Culture of cell/polymer constructs for 12 weeks led to formation of a three-dimensional tissue with architecture similar to that of native bone. These studies demonstrate that osteoblasts within a three-dimensional engineered tissue follow the classic differentiation pathway described for two-dimensional culture. Polymer scaffolds such as these may ultimately be used clinically to enhance bone regeneration by delivering or recruiting progenitor cells to the wound site.

Engineering new tissues using cell transplantation may provide a valuable tool for reconstructive surgery applications. Chondrocyte transplantation in particular has been successfully used to engineer new tissue masses due to the low metabolic requirements of these cells. However, the engineered cartilaginous tissue is too rigid for many soft tissue applications. We propose that hybrid tissue engineered from chondrocytes and smooth muscle cells could reflect mechanical properties intermediate between these two cell types. In this study, rat aortic smooth muscle cells and pig auricular chondrocytes were co-cultured on polyglycolic acid fiber-based matrices to address this hypothesis. Mixed cell suspensions were seeded by agitating the polymer matrices and a cell suspension with an orbital shaker. After seeding, cell-polymer constructs were cultured in stirred bioreactors for 8 weeks. The cell density and extracellular matrix (collagen, elastin, and glycosaminoglycan) content of the engineered tissues were determined biochemically. After 8 weeks in culture, the hybrid tissue had a high cell density (5.8 x 108 cells/cm(3)), and elastin (519 microg/g wet tissue sample), collagen (272 microg/g wet tissue sample), and glycosaminoglycan (GAG; 10 microg/g wet tissue sample) content. Mechanical testing indicated the compressive modulus of the hybrid tissues after 8 weeks to be 40.8 +/- 4.1 kPa and the equilibrium compressive modulus to be 8.4 +/- 0.8 kPa. Thus, these hybrid tissues exhibited intermediate stiffness; they were less stiff than native cartilage but stiffer than native smooth muscle tissue. This tissue engineering approach may be useful to engineer tissues for a variety of reconstructive surgery applications.

The purpose of this study was to examine the growth and morphology of a salivary epithelial cell line (HSG) in vitro on several biodegradable substrata as an important step toward developing an artificial salivary gland. The substrates examined were poly-L-lactic acid (PLLA), polyglycolic acid (PGA), and two co-polymers, 85% and 50% PLGA, respectively. The substrates were formed into 20- to 25-mm disks, and the cells were seeded directly onto the polymers or onto polymers coated with specific extracellular matrix proteins. The two copolymer substrates became friable over time in aqueous media and proved not useful for these experiments. The purified matrix proteins examined included fibronectin (FN), laminin (LN), collagen I, collagen IV, and gelatin. In the absence of preadsorbed proteins, HSG cells did not attach to the polymer disks. The cells, in general, behaved similarly on both PLLA and PGA, although optimal results were obtained consistently in PLLA. On FN-coated PLLA disks, HSG cells were able to form a uniform monolayer, which was dependent on time and FN concentration. Coating of disks with LN, collagen I, and gelatin also promoted monolayer growth. This study defines the conditions necessary for establishing a monolayer organization of salivary epithelial cells with rapid proliferation on a biodegradable substrate useful for tissue engineering.

Our laboratory has investigated hepatocyte transplantation using biodegradable polymer matrices as an alternative treatment to end-stage liver disease. One of the major limitations has been the insufficient survival of an adequate mass of transplanted cells. This study investigates a novel method of dynamic seeding and culture of hepatocytes in a flow perfusion system. In experiment I, hepatocytes were flow-seeded onto PGA scaffolds and cultured in a flow perfusion system for 24 h. Overall metabolic activity and distribution of cells were assessed by their ability to reduce MTT. DNA quantification was used to determine the number of cells attached. Culture medium was analyzed for albumin content. In Experiment II, hepatocyte/polymer constructs were cultured in a perfusion system for 2 and 7 days. The constructs were examined by SEM and histology. Culture medium was analyzed for albumin. In experiment I, an average of 4.4 X 10(6) cells attached to the scaffolds by DNA quantification. Cells maintained a high metabolic activity and secreted albumin at a rate of 13 pg/cell/day. In experiment II, SEM demonstrated successful attachment of hepatocytes on the scaffolds after 2 and 7 days. Cells appeared healthy on histology and maintained a high rate of albumin secretion through day 7. Hepatocytes can be dynamically seeded onto biodegradable polymers and survive with a high rate of albumin synthesis in the flow perfusion culture system.

The purpose of this study was to demonstrate the feasibility of end-to-end anastomosis between tissue-engineered intestine and native small bowel and to investigate the effect of this anastomosis on their growth. Microporous biodegradable polymer tubes were created from a fiber mesh of polyglycolic acid sprayed with 5% polylactic acid. Intestinal epithelial organoid units were harvested from neonatal Lewis rats and seeded onto polymers. These constructs were implanted into the omentum of adult Lewis rats. Three weeks after the implantation, the constructs (n = 7) were anastomosed to the native jejunum in an end-to-end fashion. Ten weeks after implantation, the tissue-engineered intestine was harvested. Four of 7 rats survived for 10 weeks and the overall patency rate of the anastomosis was 78% (11 of 14 anastomosis). The maximal length of the tissue-engineered intestine at week 3 and 10 was 1.80 +/- 0.32 and 1.93 +/- 0.39 cm (mean +/- SD). Histologically, the tissue-engineered intestine was lined with a well-developed neomucosal layer that was continuous with the native intestine. We conclude that anastomosis between tissue-engineered intestine and native small bowel had a moderately high patency rate and had a positive effect on maintenance of the size of the neointestine and development of the neomucosa.

Polymers of lactic and glycolic acid are attractive candidates to fabricate devides to transplant cells and engineer new tissues. These polymers are biocompatible, and exhibit a wide range of erosion times and mechanical properties. This manuscript describes the fabrication and characterization, in vitro and in vivo, of hollow, tubular devices from porous films of various polymers of this family. Porous films of these polymers were formed using a particulate leaching technique, and sealed around Teflon cylinders to form hollow tubular devices. The erosion rate of devices was controlled by the specific polymer utilized for fabrication, and ranged from months to years. Devices fabricated from a 50/50 copolymer of D,L-lactic acid and glycolic acid were completely eroded by 2 months, while devices fabricated from a homopolymer of L-lactic acid showed little mass loss after 1 year. Erosion times for devices fabricated from the other polymers [poly-(D,L-lactic acid) and a 85/15 copolymer] were between these two extremes. Devices were capable of resisting significant compressional forces (150 raN) in vitro, and the compression resistance was controlled by the polymer utilized to fabricate the devices. The ability of the devices to maintain their structure after implantation into the mesentery or omentum of laboratory rats was also dependent of the specific polymer utilized to fabricate the device. These results indicate that it is possible to fabricate tubular devices for tissue engineering applications that exhibit a wide range of erosion rates and mechanical properties.