Biomedical subjects

P X Ma

Publications and source records attributed to P X Ma.

At least 19 recordsLinked to original sources

PubMed2001

Microtubular architecture of biodegradable polymer scaffolds.

It is a relatively new approach to generate tissues with mammalian cells and scaffolds (temporary synthetic extracellular matrices). Many tissues, such as nerve, muscle, tendon, ligament, blood vessel, bone, and teeth, have tubular or fibrous bundle architectures and anisotropic properties. In this work, we have designed and fabricated highly porous scaffolds from biodegradable polymers with a novel phase-separation technique to generate controllable parallel array of microtubular architecture. Porosity as high as 97% has been achieved. The porosity, diameter of the microtubules, the tubular morphology, and their orientation are controlled by the polymer concentration, solvent system, and temperature gradient. The mechanical properties of these scaffolds are anisotropic. Osteoprogenitor cells are seeded in these three-dimensional scaffolds and cultured in vitro. The cell distribution and the neo-tissue organization are guided by the microtubular architecture. The fabrication technique can be applied to a variety of polymers, therefore the degradation rate and cell--matrix interactions can be controlled by the chemical composition of the polymers and the incorporation of bioactive moieties. These microtubular scaffolds may be used to engineer a variety of tissues with anisotropic architecture and properties.

Animals↗

PubMed2001

Engineering new bone tissue in vitro on highly porous poly(alpha-hydroxyl acids)/hydroxyapatite composite scaffolds.

Engineering new bone tissue with cells and a synthetic extracellular matrix (scaffolding) represents a new approach for the regeneration of mineralized tissues compared with the transplantation of bone (autografts or allografts). In the present work, highly porous poly(L-lactic acid) (PLLA) and PLLA/hydroxyapatite (HAP) composite scaffolds were prepared with a thermally induced phase separation technique. The scaffolds were seeded with osteoblastic cells and cultured in vitro. In the pure PLLA scaffolds, the osteoblasts attached primarily on the outer surface of the polymer. In contrast, the osteoblasts penetrated deep into the PLLA/HAP scaffolds and were uniformly distributed. The osteoblast survival percentage in the PLLA/HAP scaffolds was superior to that in the PLLA scaffolds. The osteoblasts proliferated in both types of the scaffolds, but the cell number was always higher in the PLLA/HAP composite scaffolds during 6 weeks of in vitro cultivation. Bone-specific markers (mRNAs encoding bone sialoprotein and osteocalcin) were expressed more abundantly in the PLLA/HAP composite scaffolds than in the PLLA scaffolds. The new tissue increased continuously in the PLLA/HAP composite scaffolds, whereas new tissue formed only near the surface of pure PLLA scaffolds. These results demonstrate that HAP imparts osteoconductivity and the highly porous PLLA/HAP composite scaffolds are superior to pure PLLA scaffolds for bone tissue engineering.

3T3 Cells↗

PubMed2001

Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part 1. Structure, gelation rate and mechanical properties.

Alginate gels have been used in both drug delivery and cell encapsulation applications in the bead form usually produced by dripping alginate solution into a CaCl2 bath. The major disadvantages to these systems are that the gelation rate is hard to control; the resulting structure is not uniform; and mechanically strong and complex-shaped 3-D structures are difficult to achieve. In this work controlled gelation rate was achieved with CaCO3-GDL and CaSO4-CaCO3-GDL systems, and homogeneous alginate gels were formulated as scaffolds with defined dimensions for tissue engineering applications. Gelation rate increased with increasing total calcium content, increasing proportion of CaSO4, increasing temperature and decreasing alginate concentration. Mechanical properties of the alginate gels were controlled by the compositional variables. Slower gelation systems generate more uniform and mechanically stronger gels than faster gelation systems. The compressive modulus and strength increased with alginate concentration, total calcium content, molecular weight and guluronic acid (G) content of the alginate. MC3T3-E1 osteoblastic cells were uniformly incorporated in the alginate gels and cultured in vitro. These results demonstrated how alginate gel and gel/cell systems could be formulated with controlled structure, gelation rate, and mechanical properties for tissue engineering and other biomedical applications.

3T3 Cells↗

PubMed2001

Biodegradable polymer scaffolds with well-defined interconnected spherical pore network.

Scaffolding plays pivotal role in tissue engineering. In this work, a novel processing technique has been developed to create three-dimensional biodegradable polymer scaffolds with well-controlled interconnected spherical pores. Paraffin spheres were fabricated with a dispersion method, and were bonded together through a heat treatment to form a three-dimensional assembly in a mold. Biodegradable polymers such as PLLA and PLGA were dissolved in a solvent and cast onto the paraffin sphere assembly. After dissolving the paraffin, a porous polymer scaffold was formed. The fabrication parameters were studied in relation to the pore shape, interpore connectivity, pore wall morphology, and mechanical properties of the polymer scaffolds. The compressive modulus of the scaffolds decreased with increasing porosity. Longer heat treatment time of the paraffin spheres resulted in larger openings between the pores of the scaffolds. Foams of smaller pore size (100-200 microm) resulted in significantly lower compressive modulus than that of larger pore sizes (250-350 or 420-500 microm). The PLLA foams had a skeletal structure consisting of small platelets, whereas PLGA foams had homogeneous skeletal structure. The new processing technique can tailor the polymer scaffolds for a variety of potential tissue engineering applications because of the well-controlled architecture, interpore connectivity, and mechanical properties.

Absorbable Implants↗

PubMed2001

Optimization of hepatocyte spheroid formation for hepatic tissue engineering on three-dimensional biodegradable polymer within a flow bioreactor prior to implantation.

We hypothesize that in vitro conditioning of hepatocytes within biodegradable poly-L-lactic acid (PLLA) polymer matrices prior to implantation may increase hepatocyte survival and function after transplantation. The purpose of this study was to optimize the culture conditions of hepatocytes in a pulsatile flow bioreactor. PLLA discs were seeded with rat hepatocytes in a concentration of 2.5, 5, 10, 20 and 40 x 10(6) cells/ml. Seeded discs were exposed to recirculated perpendicular flow of 0, 7, 15, 24, 32, 52 ml/min of supplemented Williams' Medium E and harvested after 6 days in flow culture. Only under flow conditions the hepatocytes formed spheroidal aggregates (SphA) of 50-260 microm in diameter with a liver-like morphology and active metabolic function. The number of SphA was examined by phase contrast microscopy and the reductive enzyme function of the hepatocytes was tested using MTT. Hematoxylin and eosin histology showed vital hepatocytes within the SphA less than 200 microm in diameter but central necrosis in the SphA exceeding this size. Immunohistochemical staining confirmed albumin production of hepatocytes within the SphA. The optimal cell seeding concentration was 10 x 10(6) cells/ml with a flow speed of 24 ml/min. SphA of hepatocytes cultured with this flow bioreactor method may prove useful as a functional unit for tissue engineering of an in vivo liver substitute.

Albumins↗

PubMed2001

Hepatic tissue engineering on 3-dimensional biodegradable polymers within a pulsatile flow bioreactor.

BACKGROUND: An optimal method for hepatocyte transplantation is not yet determined. With the principles of tissue engineering in vitro conditioning of hepatocytes on biodegradable polymer in a flow bioreactor before implantation forming spheroids may achieve increased cell mass and function to replace lost organ function in vivo. METHODS: Biodegradable poly-L-lactic (PLLA) polymer discs were seeded with rat hepatocytes in a concentration of 10 x 10(6) cells per ml and exposed to a medium flow of 24 ml/min for 1, 2, 4 and 6 days. The number and diameter of spheroidal aggregates was measured by phase-contrast microscopy. H&E histology was performed. Albumin production as hepatocyte specific function was determined by ELISA. RESULTS: Spheroids of viable hepatocytes of 50-200 microm in diameter were formed. Both the number and diameter of the spheroids increased during the first 2 days and then remained constant until day 6. Albumin production was maintained throughout the culture period. CONCLUSION: Short (2- 3 days) pre-transplant conditioning of hepatocytes in a flow bioreactor on biodegradable PLLA resulted in formation of spheroids with a liver-like morphology and preserved specific metabolic function. Tissue engineered hepatocyte spheroids on polymer may represent a functionally active and easy transplantable neotissue and may serve as an in vivo substitute for lost liver function.

Albumins↗

PubMed2000

Synthetic nano-fibrillar extracellular matrices with predesigned macroporous architectures.

Scaffolding plays a pivotal role in tissue engineering. To mimic the architecture of a natural extracellular matrix component-collagen, nona-fibrous matrices have been created with synthetic biodegradable polymers in our laboratory using a phase-separation technique. To improve the cell seeding, distribution, mass transport, and new tissue organization, three-dimensional macroporous architectures are built in the nano-fibrous matrices. Water-soluble porogen materials are first fabricated into three-dimensional negative replicas of the desired macroporous architectures. Polymer solutions are then cast over the porogen assemblies in a mold, and are thermally phase-separated to form nano-fibrous matrices. The porogen materials are leached out with water to finally form the synthetic nano-fibrous extracellular matrices with predesigned macroporous architectures. In this way, synthetic polymer matrices are created with architectural features at several levels, including the anatomical shape of the matrix, macroporous elements (100 microm to millimeters), interfiber distance (microns), and the diameter of the fibers (50-500 nm). These scaffolding materials circumvent the concerns of pathogen transmission and immuno-rejection associated with natural collagen. With the flexibility in the design of chemical structure, molecular weight, architecture, degradation rate, and mechanical properties, these novel synthetic matrices may serve as superior scaffolding for tissue engineering.

Artificial Organs↗

PubMed1999

Porous poly(L-lactic acid)/apatite composites created by biomimetic process.

Highly porous poly(L-lactic acid)/apatite composites were prepared through in situ formation of carbonated apatite onto poly(L-lactic acid) foams in a simulated body fluid. The highly porous polymer foams (up to 95% porosity) were prepared from polymer solution by solid-liquid phase separation and subsequent sublimation of the solvent. The foams were then immersed in the simulated body fluid at 37 degrees C to allow the in situ apatite formation. After incubation in the simulated body fluid for a certain period of time, a large number of characteristic microparticles formed on the surfaces of pore walls throughout the polymer foams. The microparticles were characterized with scanning electron microscopy, energy dispersive spectroscopy, Fourier transform IR spectroscopy, and X-ray diffractometry. These porous spherical microparticles were assemblies of microflakes. They were found to be carbonated bonelike apatite. A series of composite foams with varying sizes and concentrations of the apatite particles was obtained by varying incubation time and conditions. These porous composites may be promising scaffolding materials for bone tissue engineering and regeneration because the excellent bone-bonding properties of the apatite may provide a good environment for osteoblast and osteoprogenitor cells' attachment and growth.

Apatites↗

PubMed1999

Poly(alpha-hydroxyl acids)/hydroxyapatite porous composites for bone-tissue engineering. I. Preparation and morphology.

Tissue engineering has shown great promise for creating biological alternatives for implants. In this approach, scaffolding plays a pivotal role. Hydroxyapatite mimics the natural bone mineral and has shown good bone-bonding properties. This paper describes the preparation and morphologies of three-dimensional porous composites from poly(L-lactic acid) (PLLA) or poly(D,L-lactic acid-co-glycolic acid) (PLGA) solution and hydroxyapatite (HAP). A thermally induced phase separation technique was used to create the highly porous composite scaffolds for bone-tissue engineering. Freeze drying of the phase-separated polymer/HAP/solvent mixtures produced hard and tough foams with a co-continuous structure of interconnected pores and a polymer/HAP composite skeleton. The microstructure of the pores and the walls was controlled by varying the polymer concentration, HAP content, quenching temperature, polymer, and solvent utilized. The porosity increased with decreasing polymer concentration and HAP content. Foams with porosity as high as 95% were achieved. Pore sizes ranging from several microns to a few hundred microns were obtained. The composite foams showed a significant improvement in mechanical properties over pure polymer foams. They are promising scaffolds for bone-tissue engineering.

Adhesiveness↗

PubMed1999

Morphology and mechanical function of long-term in vitro engineered cartilage.

Cartilage tissue can be engineered in vitro with articular chondrocytes and poly(glycolic acid) nonwoven scaffolds as previously shown over 12 weeks in vitro. This study addressed whether engineered cartilage would further evolve and approach natural cartilage in extracellular matrix organization and biomechanical properties, especially aggregate modulus through longer term in vitro cultivation. Cellularity, cell size, compressive modulus, and permeability of the in vitro engineered cartilage stabilized within the 12-week cultivation time and remained at the same levels as those of natural cartilage thereafter. The linear range of the stress-strain curve was from 0 to a strain value between 5 and 10% for all the engineered cartilage tissues that were in vitro cultured for longer than 2 weeks, which was the same linear range for natural cartilage. The aggregate modulus further increased from week 12 to week 20 and remained approximately the same value thereafter during a 25-week in vitro cultivation. The aggregate modulus of the engineered cartilage reached 179+/-9 kPa after 20 weeks of in vitro cultivation, which was 40% that of natural articular cartilage. To our knowledge this is the highest aggregate modulus value yet reported of any in vitro engineered cartilage tissue.

Animals↗

PubMed1999

Synthetic nano-scale fibrous extracellular matrix.

Biodegradable polymers have been widely used as scaffolding materials to regenerate new tissues. To mimic natural extracellular matrix architecture, a novel highly porous structure, which is a three-dimensional interconnected fibrous network with a fiber diameter ranging from 50 to 500 nm, has been created from biodegradable aliphatic polyesters in this work. A porosity as high as 98.5% has been achieved. These nano-fibrous matrices were prepared from the polymer solutions by a procedure involving thermally induced gelation, solvent exchange, and freeze-drying. The effects of polymer concentration, thermal annealing, solvent exchange, and freezing temperature before freeze-drying on the nano-scale structures were studied. In general, at a high gelation temperature, a platelet-like structure was formed. At a low gelation temperature, the nano-fibrous structure was formed. Under the conditions for nano-fibrous matrix formation, the average fiber diameter (160-170 nm) did not change statistically with polymer concentration or gelation temperature. The porosity decreased with polymer concentration. The mechanical properties (Young's modulus and tensile strength) increased with polymer concentration. A surface-to-volume ratio of the nano-fibrous matrices was two to three orders of magnitude higher than those of fibrous nonwoven fabrics fabricated with the textile technology or foams fabricated with a particulate-leaching technique. This synthetic analogue of natural extracellular matrix combined the advantages of synthetic biodegradable polymers and the nano-scale architecture of extracellular matrix, and may provide a better environment for cell attachment and function.

Algorithms↗

PubMed1998

Creation of viable pulmonary artery autografts through tissue engineering.

BACKGROUND: "Repair" of many congenital cardiac defects requires the use of conduits to establish right ventricle to pulmonary artery continuity. At present, available homografts or prosthetic conduits lack growth potential and can become obstructed by tissue ingrowth or calcification leading to the need for multiple conduit replacements. Tissue engineering is an approach by which cells are grown in vitro onto biodegradable polymers to construct "tissues" for implantation. A tissue engineering approach has recently been used to construct living cardiac valve leaflets from autologous cells in our laboratory. This study assesses the feasibility of a tissue engineering approach to constructing tissue-engineered "living" pulmonary artery conduits. MATERIALS AND METHODS: Ovine artery (group A, n = 4) or vein (group V, n = 3) segments were harvested, separated into individual cells, expanded in tissue culture, and seeded onto synthetic biodegradable (polyglactin/polyglycolic acid) tubular scaffolds (20 mm long x 15 mm diameter). After 7 days of in vitro culture, the autologous cell/polymer vascular constructs were used to replace a 2 cm segment of pulmonary artery in lambs (age 68.4 +/- 15.5 days, weight 18.7 +/- 2.0 kg). One other control animal received an acellular polymer tube sealed with fibrin glue without autologous cells. Animals were sacrificed at intervals of 11 to 24 weeks (mean follow-up 130.3 +/- 30.8 days, mean weight 38.9 +/- 13.0 kg) after echocardiographic and angiographic studies. Explanted tissue-engineered conduits were assayed for collagen (4-hydroxyproline) and calcium content, and a tissue deoxyribonucleic acid assay (bis-benzimide dye) was used to estimate number of cell nuclei as an index of tissue maturity. RESULTS: The acellular control graft developed progressive obstruction and thrombosis. All seven tissue-engineered grafts were patent and demonstrated a nonaneurysmal increase in diameter (group A = 18.3 +/- 1.3 mm = 95.3% of native pulmonary artery; group V = 17.1 +/- 1.2 mm = 86.8% of native pulmonary artery). Histologically, none of the biodegradable polymer scaffold remained in any tissue-engineered graft by 11 weeks. Collagen content in tissue-engineered grafts was 73.9% +/- 8.0% of adjacent native pulmonary artery. Histologically, elastic fibers were present in the media layer of tissue-engineered vessel wall and endothelial specific factor VIII was identified on the luminal surface. Deoxyribonucleic acid assay showed a progressive decrease in numbers of cell nuclei over 11 and 24 weeks, suggesting an ongoing tissue remodeling. Calcium content of tissue-engineered grafts was elevated (group A = 7.95 +/- 5.09; group V = 13.2 +/- 5.48; native pulmonary artery = 1.2 +/- 0.8 mg/gm dry weight), but no macroscopic calcification was found. CONCLUSIONS: Living vascular grafts engineered from autologous cells and biodegradable polymers functioned well in the pulmonary circulation as a pulmonary artery replacement. They demonstrated an increase in diameter suggesting growth and development of endothelial lining and extracellular matrix, including collagen and elastic fibers. This tissue-engineering approach may ultimately allow the development of viable autologous vascular grafts for clinical use.

Animals↗

PubMed1998

Formation of spheroidal aggregates of hepatocytes on biodegradable polymers under continuous-flow bioreactor conditions.

Our laboratory has investigated heterotopic hepatocyte transplantation on biodegradable polymer matrices as an experimental treatment for end-stage liver disease. One of the limitations has been survival of sufficient cell mass after transplantation. We hypothesize that in vitro conditioning of cells within polymer matrices prior to implantation may increase hepatocyte survival and function. In this preliminary study we investigated the effect of continuous flow on hepatocytes and sinusoidal endothelial cells on poly-L-lactic acid (PLLA) discs in vitro. Highly porous PLLA discs were manufactured measuring 18 mm diameter by 1 mm thickness using previously described techniques. Hepatocytes were isolated from adult, male Lewis rats (200-300 g) using a two-step collagenase digestion. Sinusoidal endothelial cells were isolated using a two-step collagenase digestion, differential sedimentation, Percoll gradient centrifugation, and selective adherence. PLLA discs were seeded with hepatocytes alone or with co-cultures of hepatocytes and sinusoidal endothelial cells. Seeded discs were then secured within a flow bioreactor chamber and exposed to continuous flow of culture media at a rate of 20 ml/minute through the chamber. Seeded discs placed in static culture conditions served as controls. Specimens seeded with only hepatocytes were harvested at 24 hours, 48 hours, and 168 hours after seeding. Co-culture specimens were harvested after 168 hours. Specimens were viewed under phase-contrast microscopy and then formalin-fixed and prepared for histologic sectioning. Sections were stained with Hematoxylin and Eosin and then analyzed with light microscopy. Hepatocytes under flow conditions formed spheroidal aggregates of cells of 50 to 200 microns in diameter by 24 hours in culture. Hepatocytes in static conditions showed decreased aggregation of cells and spheroid formation was absent. Co-cultured specimens under flow also showed spheroid formation with endothelial cells lining the outside of hepatocyte spheroids. Co-cultured specimens in static culture showed no spheroid formation and no organization between sinusoidal endothelial cells and hepatocytes. These results suggest that continuous flow increases organization of hepatocytes cultured within biodegradable polymer matrices.

Animals↗

PubMed1997

Tissue-engineered heart valve leaflets: does cell origin affect outcome?

BACKGROUND: We previously reported the successful creation of tissue-engineered valve leaflet constructs and the implantation of these autologous tissue leaflets in the pulmonary valve position in a lamb model. The optimal cell origin for creating these valve leaflets remains unclear. This study was designed to compare dermal with arterial wall myofibroblasts as the cells of origin for the leaflet constructs. METHODS AND RESULTS: Mixed cell populations of endothelial cells and fibroblasts were isolated from ovine femoral arteries or subdermis and then expanded in vitro. A synthetic biodegradable polymer scaffold was then seeded with the cultured cells. The tissue scaffold was composed of a polyglactin woven mesh sandwiched between two nonwoven polyglycolic acid mesh sheets, which measured 3x3 cm in size and 3.2 mm in thickness. The cell-seeded polymer construct was implanted to replace one pulmonary valve leaflet in the same juvenile animal from which the cells had originally been obtained. Using cardiopulmonary bypass, the right posterior leaflet of the pulmonary valve was completely resected and replaced with an autologous engineered valve leaflet. In group D (n=5), the cells were obtained from subdermis, and in group A (n=4), they were obtained from the arterial wall. Eight to 10 weeks after leaflet implantation, the animals were killed, and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. The dimensions of each tissue-engineered leaflet (TEL) were compared with those of the two remaining native valve leaflets to obtain a growth index. A 4-hydroxyproline assay was performed to evaluate collagen content. Leaflet tensile strength was evaluated in vitro by using a Vitrodyne V-1000 mechanical tester. Factor VIII and elastin stains were performed to histologically assess the presence of endothelial cells and elastin, respectively. In all animals, the TEL persisted in the pulmonary valve position after 8 to 10 weeks, and all polyglycolic acid polymer had been degraded. Group A leaflets had a higher growth index (0.86+/-0.11) than group D (0.41+/-0.08) (P<.05). Macroscopically, the group D leaflets appeared thicker and contracted. Histologically, elastic fibers were more abundant in group A than in group D. Total collagen content and biomechanical testing showed no differences between groups. Leaflets from both groups had positive staining for factor VIII on the surface, confirming growth of endothelial cells to cover the TEL. CONCLUSIONS: Autologous TEL derived from vascular fibroblasts seem to develop functionally and morphologically like the native valve leaflets in the pulmonary circulation. Use of arterial myofibroblasts for the creation of TEL seems preferable to dermal fibroblasts with current tissue culture conditions.

Animals↗

PubMed1997

The effect of donor and recipient age on engraftment of tissue-engineered liver.

A novel treatment for end-stage liver disease using heterotopic hepatocyte transplantation on biodegradable polymers has been investigated. Survival and repopulation of adequate cell mass to replace hepatic function has been the principal difficulty of this method. Hence the authors have begun to investigate the role of donor and recipient age on the efficiency of hepatocyte transplantation. Lewis rats were used as donors and recipients. Hepatocytes were isolated with a collagenase digestion, both for the adult and fetal livers (17 days estimated gestational age). After digestion, the hepatocytes were seeded onto 95% porous poly-(L)-lactic acid matrices. The polymer-cell constructs with adult or fetal cells were then implanted between mesenteric leaves of three different recipient groups: adults (approximately 200 g), 2-week, and 4-week neonates (two to five animals per group, depending on litter size). The specimens were harvested at 4 weeks, stained with Hematoxylin and Eosin (H&E), and the cell area of each specimen (24 sections per group) was quantitated using morphometric analysis. Results were statistically analyzed using an unpaired, two-tailed Student's t test. At 4 weeks, all specimens showed survival of groups of hepatocytes, especially along the periphery of the polymers and near blood vessels. The hepatocyte cell area for the six groups was calculated in square micrometers: the adult cells transplanted into adult recipients, 0.16 x 10(5) microns2; fetal cells into adults, 0.47 x 10(5) microns2; adult into 4-week neonates, 1.17 x 10(5) microns2; fetal into 4-week neonates, 4.54 x 10(5) microns2; adult into 2-week neonates, 2.98 x 10(5) microns2, and fetal into 2-week neonates, 5.81 x 10(5) microns2. In all three recipient groups, the area of fetal hepatocytes was approximately two to three times the area of the adult hepatocytes (P < .05 for 2-week and 4-week neonatal recipients, P = .06 for adult recipients). Also, as the recipient age decreased, there was an increase in the hepatocyte cell area (P < .05 for fetal or adult groups). The authors conclude that fetal hepatocytes heterotopically transplanted have a significant survival advantage over adult hepatocytes, independent of recipient age. The authors further conclude that the neonatal environment is more favorable than the adult environment for implantation of hepatocytes.

Age Factors↗

PubMed1997

The in vitro construction of a tissue engineered bioprosthetic heart valve.

PROBLEM: Heart valve replacement with either a nonliving xenograft or a mechanical prosthesis is an effective therapy for valvular heart disease. Both of these approaches have limitations, including their inability to grow, repair, and remodel. In addition, a mechanical prosthesis requires long-term anticoagulation therapy. METHODS: This study demonstrates the in vitro creation of tissue engineered heart valve tissue using cardiovascular cells on degradable polymer matrices, 40 heart valve leaflets were created using this technique from two sources. Xenograft leaflets were created using human dermal fibroblasts and bovine aortic endothelial cells (n = 20) or allograft valve leaflets were created using sheep myofibroblasts and sheep endothelial cells (n = 20). A mixed sheep cell population was obtained consisting of endothelial cells and myofibroblasts. Endothelial cells were labelled with acethylated low density lipoprotein (Ac-Dil-LDL) and cells were separated into two groups using an activated cell sorter: LDL positive cells comprised of a pure endothelial cell population and LDL negative cells comprised of mixed cell population containing myofibroblasts and smooth muscle cells. The LDL negative cells were seeded on a synthetic polyglycolic acid (PGA) mesh and grown in vitro to form a tissue-like fibroblast-mesh core. Endothelial cells were then seeded onto the surface of the fibroblast-mesh core, forming a single monolayer. RESULTS: Histological evaluation of these constructs revealed an inner core of LDL negative cells and outer endothelial-like cells which were factor VIII positive. There was no evidence of capillary formation from endothelial cells invading the myofibroblasts and smooth muscle matrix and the endothelial lining appeared complete. CONCLUSIONS: It is feasible to construct allogenic heart valve tissue which could be used to make a valve.

Animals↗

PubMed1996

Tissue-engineered heart valves. Autologous valve leaflet replacement study in a lamb model.

BACKGROUND: We have previously reported the successful creation of tissue-engineered valve leaflets and the implantation of these autologous tissue leaflets in the pulmonary valve position. This study was designed to trace cultured cells that were seeded onto a biodegradable polymer with the use of a 1,1'-dioctadecyl-3,3,3' 3'-tetramethylindo-carbocyanine perchlorate (Di-1) cell-labeling method. We also examined the time-related biochemical, biomechanical, and histological characteristics and evolution of these tissue constructs. METHODS AND RESULTS: Mixed cell populations of endothelial cells and fibroblasts were isolated from explanted ovine arteries. Endothelial cells were selectively labeled with an acetylated low density lipoprotein marker and separated from fibroblasts with the use of a fluorescence-activated cell sorter. A synthetic biodegradable scaffold consisting of polyglycolic acid fibers was seeded first with fibroblasts, then coated with endothelial cells. Using these methods, we implanted autologous cell/polymer constructs in six animals. In two additional control animals, a leaflet of polymer was implanted without prior cell seeding. In each animal, cardiopulmonary bypass was used to completely resect the right posterior leaflet of the pulmonary valve and replace it with an engineered valve leaflet with (n = 6) or without (n = 2) prior cultured cell seeding. The animals were killed either after 6 hours or after 1, 6, 7, 9, or 11 weeks, and the implanted valve leaflets were examined histologically, biochemically, and biomechanically. 4-Hydroxyproline assays were performed to determine collagen content. Leaflet strength was evaluated in vitro with a mechanical tester Factor VIII and elastin stains were done to verify histologically that endothelial cells and elastin, respectively, were present. Animals receiving leaflets made from polymers without cell seeding were killed and examined in a similar fashion after 8 weeks. In the control animals, the acellular polymer leaflets were completely degraded, with no residual leaflet tissue at 8 weeks. The tissue-engineered valve leaflet persisted in each animal in the experimental group. 4-Hydroxyproline analysis of the constructs showed a progressive increase in collagen content. Immunohistochemical staining demonstrated elastin fibers in the matrix and factor VIII on the surface of the leaflet. The cell-labeling experiments demonstrated that the cells on the leaflets had persisted from the in vitro seeding of the leaflets. CONCLUSIONS: In the tissue-engineered heart valve leaflet, transplanted autologous cells generated a proper matrix on the polymer scaffold in a physiological environment at a period of 8 weeks after implantation.

Animals↗

PubMed1995

Development of biomechanical properties and morphogenesis of in vitro tissue engineered cartilage.

Neocartilage was engineered by culturing bovine chondrocytes on poly(glycolic acid) (PGA) fibrous nonwoven scaffolds. The biomechanical properties and morphologies of the PGA-chondrocyte constructs were studied over 12 weeks of in vitro culture. PGA scaffolds without cells lost their mechanical strength and structural integrity between week 2 and week 3 in culture. The thickness of the PGA-chondrocyte constructs decreased by 35% during the first 3 weeks, but the thickness increased from week 3 to week 9 to a thickness 42% higher than that of the starting scaffolds, which was then maintained. Safranin O staining of PGA-chondrocyte constructs revealed increasing proteoglycan formation over time. The compressive modules of PGA-chondrocyte constructs increased with in vitro culture time, and reached the same order of magnitude as that of normal bovine cartilage at week 9. The aggregate modulus of the PGA-chondrocyte constructs decreased by 57% over the first 2 weeks but then increased, reaching the same order of magnitude as normal bovine cartilage at week 12. The apparent permeability of the PGA-chondrocyte constructs, which was initially four orders of magnitude above that of normal cartilage, decreased between weeks 1 and 3 and thereafter remained the same order of magnitude as that measured for normal cartilage.

Animals↗