Biomedical subjects

Amit K Roy

Publications and source records attributed to Amit K Roy.

10 recordsLinked to original sources

PubMed2006

Tissue-engineered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth.

In this study, we describe the isolation and characterization of a population of adult-derived or somatic lung progenitor cells (SLPC) from adult mammalian lung tissue and the promotion of alveolar tissue growth by these cells (both in vitro and in vivo) after seeding onto synthetic polymer scaffolds. After extended in vitro culture, differentiating cells expressed Clara cell 10kDa protein, surfactant protein-C, and cytokeratin but did not form organized structures. When cells were combined with synthetic scaffolds, polyglycolic acid (PGA) or Pluronic F-127 (PF-127), and maintained in vitro or implanted in vivo, they expressed lung-specific markers for Clara cells, pneumocytes, and respiratory epithelium and organized into identifiable pulmonary structures (including those similar to alveoli and terminal bronchi), with evidence of smooth muscle development. Although PGA has been shown to be an excellent polymer for culture of specific cell types in vitro, in vivo culture in an immunocompetent host induced a foreign body response that altered the integrity of the developing lung tissue. Use of PF-127/cell constructs resulted in the development of tissue with less inflammatory reaction. These data suggest that the therapeutic use of engineered tissues requires both the use of specific cell phenotypes, as well as the careful selection of synthetic polymers, to facilitate the assembly of functional tissue.

Animals↗

PubMed2005

Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs.

Composite tissue-engineered intervertebral tissue was assembled in the shape of cylindrical disks composed of an outer shell of PGA mesh seeded with annulus fibrosus cells with an inner core of nucleus pulposus cells seeded into an alginate gel. Samples were implanted subcutaneously in athymic mice and retrieved at time points up to 16 weeks. At all retrieval times, samples maintained shape and contained regions of distinct tissue formation. Histology revealed progressive tissue formation with distinct morphological differences in tissue formation in regions seeded with annulus fibrosus and nucleus pulposus cells. Biochemical analysis indicated that DNA, proteoglycan, and collagen content in tissue-engineered discs increased with time, reaching >50% of the levels of native tissue by 16 weeks. The exception to this was the collagen content of the nucleus pulposus portion of the implants with were approximately 15% of native values. The equilibrium modulus of tissue-engineered discs was 49.0+/-13.2 kPa at 16 weeks, which was between the measured values for the modulus of annulus fibrosus and nucleus pulposus. The hydraulic permeability of tissue-engineered discs was 5.1+/-1.7x10(-14) m2/Pa at 16 weeks, which was between the measured values for the hydraulic permeability of annulus fibrosus and nucleus pulposus. These studies document the feasibility of creating composite tissue-engineered intevertebral disc implants with similar composition and mechanical properties to native tissue.

Alginates↗

PubMed2004

Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement.

STUDY DESIGN: By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks. OBJECTIVES: The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively. SUMMARY OF BACKGROUND DATA: Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants. METHODS: Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in -70 degrees C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining. RESULTS: The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc. CONCLUSION: These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

Animals↗

PubMed2004

Injectable tissue-engineered cartilage with different chondrocyte sources.

Injectable engineered cartilage that maintains a predictable shape and volume would allow recontouring of craniomaxillofacial irregularities with minimally invasive techniques. This study investigated how chondrocytes from different cartilage sources, encapsulated in fibrin polymer, affected construct mass and volume with time. Swine auricular, costal, and articular chondrocytes were isolated and mixed with fibrin polymer (cell concentration of 40 x 10 cells/ml for all groups). Eight samples (1 cm x 1 cm x 0.3 cm) per group were implanted into nude mice for each time period (4, 8, and 12 weeks). The dimensions and mass of each specimen were recorded before implantation and after explantation. Ratios comparing final measurements and original measurements were calculated. Histological, biochemical, and biomechanical analyses were performed. Histological evaluations (n = 3) indicated that new cartilaginous matrix was synthesized by the transplanted chondrocytes in all experimental groups. At 12 weeks, the ratios of dimension and mass (n = 8) for auricular chondrocyte constructs increased by 20 to 30 percent, the ratios for costal chondrocyte constructs were equal to the initial values, and the ratios for articular chondrocyte constructs decreased by 40 to 50 percent. Constructs made with auricular chondrocytes had the highest modulus (n = 3 to 5) and glycosaminoglycan content (n = 4 or 5) and the lowest permeability value (n = 3 to 5) and water content (n = 4 or 5). Constructs made with articular chondrocytes had the lowest modulus and glycosaminoglycan content and the highest permeability value and water content (p < 0.05). The amounts of hydroxyproline (n = 5) and DNA (n = 5) were not significantly different among the experimental groups (p > 0.05). It was possible to engineer injectable cartilage with chondrocytes from different sources, resulting in neocartilage with different properties. Although cartilage made with articular chondrocytes shrank and cartilage made with auricular chondrocytes overgrew, the injectable tissue-engineered cartilage made with costal chondrocytes was stable during the time periods studied. Furthermore, the biomechanical properties of the engineered cartilage made with auricular or costal chondrocytes were superior to those of cartilage made with articular chondrocytes, in this model.

Animals↗

PubMed2003

A composite tissue-engineered trachea using sheep nasal chondrocyte and epithelial cells.

This study evaluates the feasibility of producing a composite engineered tracheal equivalent composed of cylindrical cartilaginous structures with lumens lined with nasal epithelial cells. Chondrocytes and epithelial cells isolated from sheep nasal septum were cultured in Ham's F12 media. After 2 wk, chondrocyte suspensions were seeded onto a matrix of polyglycolic acid. Cell-polymer constructs were wrapped around silicon tubes and cultured in vitro for 1 wk, followed by implanting into subcutaneous pockets on the backs of nude mice. After 6 wk, epithelial cells were suspended in a hydrogel and injected into the embedded cartilaginous cylinders following removal of the silicon tube. Implants were harvested 4 wk later and analyzed. The morphology of implants resembles that of native sheep trachea. H&E staining shows the presence of mature cartilage and formation of a pseudo-stratified columnar epithelium, with a distinct interface between tissue-engineered cartilage and epithelium. Safranin-O staining shows that tissue-engineered cartilage is organized into lobules with round, angular lacunae, each containing a single chondrocyte. Proteoglycan and hydroxyproline contents are similar to native cartilage. This study demonstrates the feasibility of recreating the cartilage and epithelial portion of the trachea using tissue harvested in a single procedure. This has the potential to facilitate an autologous repair of segmental tracheal defects.

Animals↗

PubMed2003

Tissue engineering of autologous cartilage for craniofacial reconstruction by injection molding.

Each year, more than one million patients undergo some type of procedure involving cartilage reconstruction. Polymer hydrogels such as alginate have been demonstrated to be effective carriers of chondrocytes for subcutaneous cartilage formation. The goal of this study was to develop a simple method to create complex structures with good three-dimensional tolerance in order to form cartilage in specific shapes in an autologous animal model. Six alginate implants that had been seeded with autologous chondrocytes through an injection molding process were implanted subcutaneously in sheep, harvested after 6 months, and analyzed histologically, biochemically, and biomechanically, in comparison with original auricular cartilage. Molds of craniofacial implants were prepared with Silastic E RTV (Dow Corning, Midland, Mich.). Chondrocytes were harvested from sheep auricular cartilage and suspended in 2% alginate at a concentration of 50 x 10(6) cells/ml. The mixture of cells and gel was injected into the Silastic molds and removed after 20 minutes. Chondrocyte-alginate constructs were implanted subcutaneously in the necks of the sheep from which the cells had originally been harvested, and the constructs were removed after 30 weeks. Analyses of the implanted constructs indicated cartilage formation with three-dimensional shape retention. The proteoglycan and collagen contents of the constructs increased with time to approximately 80 percent of the values for native tissue. The equilibrium modulus and the hydraulic permeability were 74 and 105 percent of those of native sheep auricular cartilage, respectively.

Alginates↗

PubMed2002

Age-related changes in the composition and mechanical properties of human nasal cartilage.

Nasal cartilage is widely used in reconstructive surgery for the replacement of soft tissue defects and nasal reconstruction procedures. The ability to shape harvested tissue and the performance in the transplant site are related to the mechanical properties of nasal cartilage. Several studies have documented changes in composition and mechanical properties of other cartilages with age, but little is known about these processes in nasal cartilage. In this study, 45 human nasal septum specimens were gathered from patients 15-60 years of age after reconstructive surgery. Samples were cut to 6 mm in diameter and tested in confined compression to determine equilibrium modulus and hydraulic permeability and analyzed for glycosaminoglycan and hydroxyproline content. Equilibrium modulus decreased significantly with increasing donor age (P<0.01) while hydraulic permeability increased significantly (P<0.02). Glycosaminoglycan (GAG) content decreased significantly with age (P<0.05), while hydroxyproline content showed a slight, but not significant, increase with age (P>0.2). These trends are qualitatively similar to those observed in articular cartilage, suggesting the existence of a systemic process of cartilage degradation that is independent of mechanical loading. Further, the relationships between biochemical composition and mechanical properties were age-dependent, with cartilage from patients less than 30 years of age showing greater dependence of equilibrium modulus and hydraulic permeability on GAG and hydroxyproline content. This suggests that changes in matrix organization may accompany changes in tissue composition.

Adolescent↗

PubMed2002

Age dependence of biochemical and biomechanical properties of tissue-engineered human septal cartilage.

The aim of this study was to determine whether the biomechanical and biochemical properties of tissue-engineered human septal cartilage vary with donor age and in vitro culture time. Chondrocytes were isolated from human septal cartilage of patients from 15 to 60 year old and maintained in primary monolayer culture for 14 days. Cells were seeded onto 0.5% PLA coated PGA disks and kept in stationary three-dimensional culture for either 1 day or 3 weeks. Specimens were then implanted subcutaneously into athymic nude mice and harvested after either 4 or 8 weeks. Upon harvest, the equilibrium confined compression modulus was measured as to quantify mechanical properties, and the glycosaminoglycan, hydroxyproline, and DNA contents were determined as measures of tissue proteoglycans, collagen, and cell density. This study demonstrated that native nasal cartilage showed distinct changes in these parameters with age, but cartilage engineered using the cells of these specimens showed no significant dependence on the age of the donor. There was little difference in quality of cartilage between samples cultured for 3 weeks in vitro and those implanted directly after seeding. Together, the results of this study suggest that the process of extracellular matrix assembly by chondrocytes on three-dimensional scaffolds may be independent of in vivo conditions experienced by the tissue prior to harvest.

Adolescent↗

PubMed2002

Autologous tissue-engineered trachea with sheep nasal chondrocytes.

OBJECTIVE: This study was designed to evaluate the ability of autologous tissue-engineered trachea shaped in a helix to form the structural component of a functional tracheal replacement. METHODS: Nasal septum were harvested from six 2-month-old sheep. Chondrocytes and fibroblasts were isolated from tissue and cultured in media for 2 weeks. Both types of cells were seeded onto separate nonwoven meshes of polyglycolic acid. The chondrocyte-seeded mesh was wound around a 20-mm-diameter x 50-mm-long helical template and then covered with the fibroblast-seeded mesh. In 2 separate studies the implants were placed either in a subcutaneous pocket in the nude rat (rat tissue-engineered trachea) or in the neck of a sheep (sheep tissue-engineered trachea). Rat tissue-engineered tracheas were harvested after 8 weeks and analyzed by means of histology and biochemistry. Sheep tissue-engineered tracheas were harvested from the neck at 8 weeks and anastomosed into a 5-cm defect in the sheep trachea. RESULTS: Sheep receiving tissue-engineered trachea grafts survived for 2 to 7 days after implantation. Gross morphology and tissue morphology were similar to that of native tracheas. Hematoxylin-and-eosin staining of rat tissue-engineered tracheas and sheep tissue-engineered tracheas revealed the presence of mature cartilage surrounded by connective tissue. Safranin-O staining showed that rat tissue-engineered tracheas and sheep tissue-engineered tracheas had similar morphologies to native tracheal cartilage. Collagen, proteoglycan, and cell contents were similar to those seen in native tracheal tissue in rat tissue-engineered tracheas. Collagen and cell contents of sheep tissue-engineered tracheas were elevated compared with that of normal tracheas, whereas proteoglycan content was less than that found in normal tracheas. CONCLUSIONS: This study demonstrated the feasibility of recreating the cartilage and fibrous portion of the trachea with autologous tissue harvested from single procedure. This approach might provide a benefit to individuals needing tracheal resection.

Animals↗

PubMedDate not supplied

Role for interleukin 1alpha in the inhibition of chondrogenesis in autologous implants using polyglycolic acid-polylactic acid scaffolds.

Significant challenges remain in generating tissue-engineered cartilage in immunocompetent animals. Scaffold materials such as polyglycolic acid lead to significant inflammatory reactions, inhibiting homogeneous matrix synthesis. This study examined the generation of tissue-engineered cartilage, using a polyglycolic acid-polylactic acid copolymer (Ethisorb; Ethicon, Norderstedt, Germany) in an autologous immunocompetent pig model. The goals of this study were to determine the role of interleukin 1alpha (IL-1alpha) in this system and to assess the effect of serum treatment on tissue generation. Porcine auricular chondrocytes were seeded onto Ethisorb disks cultured for 1 week in medium supplemented with either fetal bovine serum or serum-free insulin-transferrin-selenium supplement. Specimens were implanted autogenously in pigs with unseeded scaffolds as controls. After 1, 4, or 8 weeks, six specimens from each group were explanted and analyzed histologically (hematoxylin and eosin, safranin O, trichrome, and Verhoeff's staining) and biochemically (glycosaminoglycan content). The presence and distribution of IL-1alpha were assessed by immunohistochemistry. Histology revealed acute inflammation surrounding degrading scaffold. Cartilage formation was observed as early as 1 week after implantation and continued to increase with time; however, homogeneous matrix synthesis was not present in any of the specimens. Strong IL-1alpha expression was detected in chondrocytes at the implant periphery and in cells in the vicinity of degrading polymer. Histologically there was no significant difference between the experimental groups with respect to the amount of matrix synthesis or inflammatory infiltration. The glycosaminoglycan content was significantly higher in the serum-free group. These results suggest that inflammatory reactions against scaffold materials and serum components lead to the production of cytokines such as IL-1alpha that may inhibit cartilage tissue formation in autologous transplant models.

Absorbable Implants↗