Amit K. Roy

Injection molding of chondrocyte/alginate constructs in the shape of facial implants

Over one million patients per year undergo some type of procedure involving cartilage reconstruction. Polymer hydrogels, such as alginate, have been shown to be effective carriers for chondrocytes in subcutaneous cartilage formation. The goal of our current study was to develop a method to create complex structures (nose bridge, chin, etc.) with good dimensional tolerance to form cartilage in specific shapes. Molds of facial implants were prepared using Silastic ERTV. Suspensions of chondrocytes in 2% alginate were gelled by mixing with CaSO(4) (0.2 g/mL) and injected into the molds. Constructs of various cell concentrations (10, 25, and 50 million/mL) were implanted in the dorsal aspect of nude mice and harvested at times up to 30 weeks. Analysis of implanted constructs indicated progressive cartilage formation with time. Proteoglycan and collagen constructs increased with time to approximately 60% that of native tissue. Equilibrium modulus likewise increased with time to 15% that of normal tissue, whereas hydraulic permeability decreased to 20 times that of native tissue. Implants seeded with greater concentrations of cells increased proteoglycan content and collagen content and equilibrium and decreased permeability. Production of shaped cartilage implants by this technique presents several advantages, including good dimensional tolerance, high sample-to-sample reproducibility, and high cell viability. This system may be useful in the large-scale production of precisely shaped cartilage implants.

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 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.

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 Hams 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 pseudostratified 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.—Kojima, K., Bonassar, L. J., Roy, A. K., Mizuno, H., Cortiella, J., Vacanti, C. A. A composite tissue‐engineered trachea using sheep nasal chondrocyte and epithelial cells. FASEB J. 17, 823–828 (2003)

Identification and initial characterization of spore-like cells in adult mammals

We describe the identification and initial characterization of a novel cell type that seems to be present in all tissues. To date we have isolated what we term “spore‐like cells” based on the characteristics described below. They are extremely small, in the range of less than 5 μm, and appear to lie dormant and to be dispersed throughout the parenchyma of virtually every tissue in the body. Being dormant, they survive in extremely low oxygen environments, as evidenced by their viability in tissues (even in metabolically very active tissues such as the brain or spinal cord) for several days after sacrifice of an animal without delivery of oxygen or nutrients. The spore‐like cells described in this report have an exceptional ability to survive in hostile conditions, known to be detrimental to mammalian cells, including extremes of temperature. Spore‐like cells remain viable in unprepared tissue, frozen at −86°C (using no special preservation techniques) and then thawed, or heated to 85°C for more than 30 min. Preliminary characterization of these cells utilizing basic and special stains, as well as scanning and transmission electron microscopy reveal very small undifferentiated cells, which contain predominantly nucleus within a small amount of cytoplasm and a few mitochondria. Focal periodic acid‐Schiff and mucicarmine stains suggest a coating of glycolipid and mucopolysaccharide. In vitro, these structures have the capacity to enlarge, develop, and differentiate into cell types expressing characteristics appropriate to the tissue environment from which they were initially isolated. We believe that these unique cells lie dormant until activated by injury or disease, and that they have the potential to regenerate tissues lost to disease or damage. J. Cell. Biochem. 80:455–460, 2001.

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 × 106 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.

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.

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.