Derivation of neurospheres from bone marrow stromal cells.

Nerve stem cells have a unique characteristic in that they form spherical aggregates, also termed neurospheres, in vitro. The study demonstrated the successful derivation of these neurospheres from bone marrow culture. Their plasticity as nerve stem cells was confirmed. The findings further strengthens the pluripotency of cell populations within the bone marrow.

Composite tissue formation derived solely from a blood biological matrix: a preliminary study.

We describe the formation of a new composite tissue containing all of the cellular components of adjacent normal spinal tissue. Four millimeter gaps, surgically created at the level of T8-T9 in the spinal cord of 2 adult female Lewis rats, resulted in complete paralysis of the lower extremities. A biological matrix derived from previously frozen peripheral syngeneic blood was implanted into the created spinal cord defects in 2 experimental animals. Two control animals received fibrin implants not containing matrix material. The 2 experimental animals regained significant motor function of their lower extremities as compared with the control animals, over a period of 2 months. Histological analysis of the experimental implants showed a composite tissue formation consisting of neural tissue, bone, and cartilage similar in cellular content to the adjacent native tissue. Only organized blood clot was seen at the implant site of the control animals. Further studies are needed to determine if autologous blood can be used to regenerate tissues lost to disease or injury.

Tissue engineered cartilage: utilization of autologous serum and serum-free media for chondrocyte culture.

BACKGROUND: Standard culture medium contains bovine serum. If standard culture methodology is used for future human tissue-engineering, unknown risks of infection from bovine disease or immune reaction to foreign proteins theoretically might occur. In this study we wished to evaluate the potential of chondrocyte expansion using autologous and serum free media. METHODS: Autologous auricular cartilage was harvested in a swine model. An initial concentration of 100x10(3) cells per group were expanded in three groups. Group A, F-12 with 10% fetal calf serum; Group B, F-12 supplemented with 10% autologous serum; Group C, F-12 supplemented with growth factors. Cell numbers were counted at days 3, 6, 9 and 12. RESULTS: The cells in all the three groups exhibited normal chondrocyte morphology. At early time points there was a statistically significant difference in the number of cells between Group A and the two other groups (p<0.05). By day 12, both Groups A and C demonstrated greater cell number as compared to Group B (p<0.05). CONCLUSION: The results suggest that both autologous serum as well as serum free media might be substituted for the expansion of the number of chondrocytes, thus avoiding the potential need for a bovine serum supplement.

Tissue engineering of a human sized and shaped auricle using a mold.

OBJECTIVES: The creation of a tissue-engineered auricle was initially successful in an immunocompromised nude mouse model. Subsequently, an immunocompetent porcine model successfully generated a helical construct. We wished to evaluate the novel technique of using a mold to create a complete, anatomically refined auricle in a large animal model. METHODS: Mixtures of autogenous chondrocytes and biodegradable polymers were used inside a perforated, auricle shaped hollow gold mold. Three biodegradable polymers (calcium alginate, pluronic F-127, and polyglycolic acid) were used to retain the seeded chondrocytes inside the mold. These molds, along with a control, were implanted subcutaneously in the abdominal area of 10 animals (pigs and sheep). The constructs were removed after 8 to 20 weeks and were assessed by gross morphology and histology. RESULTS: All the gold implants were well tolerated by the animals. The implants using calcium alginate (n = 3) generated constructs of the exact shape and size of a normal human ear; the histology demonstrated mostly normal cartilage with some persistent alginate. The implants with pluronic F-127 (n = 3) resulted in cartilage with essentially normal histology, although leakage outside the molds and external cartilage generation was noted. Polyglycolic acid implants (n = 3) produced no useful cartilage because of an inflammatory reaction with fibrosis. The empty control mold (n = 1) demonstrated only a very small amount of fibrous tissue inside. CONCLUSION: A tissue-engineered human sized auricle of normal anatomic definition can be generated in an immunocompetent large-animal model using a mold technique. Although further refinements will be necessary, the technique appears promising for potential use in patients with microtia.

Expansion of the number of human auricular chondrocytes: recycling of culture media containing floating cells.

To grow a complete human size auricle by utilizing the principles of tissue engineering, a large number of chondrocytes is required for initial implantation. The number of chondrocytes can be increased by repeated passaging or by incubation with different growth factors, both of which can promote dedifferentiation. New methods of chondrocyte expansion over a relatively brief time period for potential practical application are required. In this study auricular chondrocytes were obtained from patients and cultured in vitro. Two groups of cells were created. Group A chondrocyte number was increased by repeated passaging. Group B cells were grown from floating culture medium and their number was increased both by passaging and by repeated recycling of the culture medium. Chondrocytes from both groups were implanted in nude mice for 8 weeks to generate tissue-engineered cartilage. Flow cytometry studies performed on both groups confirmed the presence of two distinct populations of structures as the source of chondrocytes from the recycled medium. Repeated recycling of the culture medium demonstrates a promising method to increase the number of chondrocytes in vitro for clinical application.

Tissue-engineered human auricular cartilage demonstrates euploidy by flow cytometry.

Transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) are known to stimulate the rate of chondrocyte proliferation. The theoretical risk of malignant transformation associated with growth factor stimulation of chondrocytes should be addressed; aneuploidy has been found to occur in human cartilaginous tumors. In this study, chondrocytes were obtained from six human auricles and cultured in vitro for 6 weeks in the presence or absence of TGF-beta and bFGF. Cells were analyzed for DNA at 3-, 4-, 5-, and 6-week intervals by flow cytometry (FACScan), which demonstrated no evidence of aneuploidy. A persistent increase in S-phase was noted in cells cultured only with TGF-beta. Cells were implanted in athymic mice, and after 8 weeks of implantation, the cartilage constructs formed were examined histologically. The tissue-engineered cartilage cultured originally in bFGF most resembled normal, native cartilage. Specimens cultured in TGF-beta produced suboptimal cartilage morphology. Flow cytometry shows no evidence of aneuploidy, with chondrocytes maintaining their normal diploid state. Further studies incorporating additional methods of analysis need to be done.

Age dependence of cellular properties of human septal cartilage: implications for tissue engineering.

BACKGROUND: The persistent need for cartilage replacement material in head and neck surgery has led to novel cell culture methods developed to engineer cartilage. Currently, there is no consensus on an optimal source of cells for these endeavors. OBJECTIVES: To evaluate human nasal cartilage as a potential source of chondrocytes and to determine the effect of donor age on cellular and proliferation characteristics. SUBJECTS: Nasal cartilage specimens were obtained after reconstructive surgery from 46 patients ranging in age from 15 to 60 years. METHODS: Specimens were weighed and chondrocytes were isolated by digestion in 0.2% collagenase type II for 16 hours. Cells were maintained in primary cultures until confluency, then seeded onto polylactic acid-polyglycolic acid scaffolds. Seeding efficiency was determined by quantification of DNA content of seeded constructs by means of Hoechst dye 33258. Specimen weights, cell yields, cell content, and doubling time were also measured and correlated to donor age. RESULTS: Mean (+/-SD) cartilage mass obtained (648 +/- 229 mg) is higher than from typical biopsy specimens of auricular cartilage, and the cellular characteristics show a higher proliferation rate than auricular chondrocytes. Cell yield increased with age, while doubling time decreased with age in samples from patients ranging from 15 to 60 years old. CONCLUSIONS: The use of nasal septal cartilage as a source of cells for tissue engineering may be valid over a wide range of patient ages. The large tissue yield and consequent cell yield make this tissue a potential starting source of chondrocytes for large-volume tissue-engineered implants.

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.

The effect of fibroblast growth factor and transforming growth factor-beta on porcine chondrocytes and tissue-engineered autologous elastic cartilage.

Elastic cartilage responds mitogenically in vitro to transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (basic FGF). We studied the effects of these growth factors separately or in a combination on porcine auricular chondrocytes in vitro and on the autologous elastic cartilage produced. Cells were harvested from the elastic auricular cartilage of 16- to 18-kg Yorkshire swine. Viability and quantification of the cells was determined. Cells were plated at equal concentration and studied in vitro in one of four identical media environments except for the growth factors: Group I contained Ham's F-12 with supplements but no growth factors, Group II also contained basic-FGF, Group III also contained TGF-beta, and Group IV also contained a combination of both growth factors. After 3 weeks in vitro, the cells were chemically dissociated with 0.25% trypsin. Cell suspensions composed of 3 x 10(7) cells/cc in 30% Pluronic F-127/Ham's F-12 were injected subcutaneously. Implants were harvested at 6, 8, 10, and 12 weeks of in vivo culture and then were examined with histologic stains. After 3 weeks of in vitro culture the total number of cells was as follows: Group I, 1.8 x 10(8); Group II, 3.5 x 10(8); Group III, 1.3 x 10(8); Group IV, 2.5 x 10(8). After 8 weeks of in vivo autologous implantation, the average weight (g) and volume (cm3) of each group was as follows: Group I, 0.7 g/0.15 cm3; Group II, 1.5 g/0.8 cm3; Group III, 0.6 g/0.1 cm3; Group IV, 1.2 g/0.3 cm3. Histologically, Groups I, II, and IV generated cartilage similar to native elastic cartilage, but Group III specimens demonstrated fibrous tissue ingrowth. Basic FGF produced the most positive enhancement on the quantity and quality of autologous tissue engineered elastic cartilage produced in this porcine model both in vitro and in vivo.

Tissue-engineered composites of bone and cartilage for mandible condylar reconstruction.

PURPOSE: This study evaluated the feasibility of creating a tissue-engineered adult human mandible condyle composite of bone and cartilage. MATERIALS AND METHODS: A polymer template composed of polyglycolic acid (PGA) and polylactic acid (PIA), and formed in the shape of the human mandible condyle, was seeded with osteoblasts isolated from a bovine periosteum suspended in calcium alginate. Chondrocytes isolated from the same calf suspended in 30% pluronic were then "painted" onto the articular surface of the scaffold, and it was then implanted into subcutaneous pockets on the dorsum of athymic mice. Animals were divided into 3 groups: group I (n = 6) received a PGA/PLA scaffold saturated with hydrogels not containing cells; group II (n = 6) received scaffolds seeded with both cell types suspended in saline rather than hydrogels; and group III (n = 6) received scaffolds seeded with both cell types suspended in hydrogel composites. Constructs were harvested after 12 weeks and evaluated grossly and microscopically by using histologic stains. RESULTS: In group I, the constructs formed a small mass without evidence of new bone or cartilage. In group II, the constructs were small and irregular. Microscopically they contained scattered islands of bone and cartilage. All specimens in group III retained their original condylar shape and were quite firm. Microscopic evaluation indicated trabecular bone interfacing with hyaline cartilage on the articulating surface. CONCLUSION: These findings show that the composites of bone and cartilage can be engineered to serve as condylar substitutes. The interdigitation of bone and cartilage at their interface is similar to the normal interface of these composite tissues seen in articulating joints.

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 microm, 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 degrees C (using no special preservation techniques) and then thawed, or heated to 85 degrees 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.

Diffusion of nitrous oxide into the pleural cavity.

We postulated that nitrous oxide transfer into the pleural cavity can occur by diffusion from the alveoli, independent of vascular transport. Under general anaesthesia, six sheep were studied in two phases, a control and an experimental phase. The sheep were anaesthetized, intubated, and received positive pressure mechanical ventilation. A catheter was placed in the right pleural cavity and 150 ml air injected. The animals were ventilated with 100% oxygen. The inspired gas was changed to a mixture of 50% nitrous oxide and 50% oxygen, and the rate of increase of nitrous oxide concentration in the pleural space was measured. The animals were then ventilated with 100% oxygen and then killed by exsanguination while ventilation was continued. The inspired mixture was changed to 50% nitrous oxide and 50% oxygen and the rate of increase in nitrous oxide concentration was measured in the pleural space again. During venitilation with nitrous oxide in the living animals, the concentration of nitrous oxide in the pleural cavity increased rapidly and decreased to zero during ventilation with 100% oxygen. During ventilation without circulation, the rate of increase in the concentration of nitrous oxide in the pleural cavity was the same as in the control phase. This suggests that nitrous oxide enters the pleural space by diffusion, rather than by vascular delivery. This mechanism may explain the rapid increase in the volume of pneumothorax if nitrous oxide is given in the inspired gas.

Internal support of tissue-engineered cartilage.

BACKGROUND: Auricles previously created by tissue engineering in nude mice used a biodegradable internal scaffold to maintain the desired shape of an ear. However, the biodegradable scaffold incited a compromising inflammatory response in subsequent experiments in immunocompetent animals. OBJECTIVE: To test the hypothesis that tissue-engineered autologous cartilage can be bioincorporated with a nonreactive, permanent endoskeletal scaffold. MATERIALS AND METHODS: Auricular elastic cartilage was harvested from Yorkshire swine. The chondrocytes were isolated and suspended into a hydrogel (Pluronic F-127) at a cell concentration of 5 x 10(7) cells/mL. Nonbiodegradable endoskeletal scaffolds were formed with 1 of 5 polymers: (1) high-density polyethylene, (2) soft acrylic, (3) polymethylmethacrylate, (4) extrapurified Silastic, and (5) conventional Silastic. Three groups were studied: (1) a control group using only the 5 polymers, (2) the 5 polymers enveloped by Pluronic F-127 only, and (3) the implants coated with Pluronic F-127 seeded with chondrocytes. All constructs were implanted subdermally; implants containing cells were implanted into the same animal from which the cells had been islolated. The implants were harvested after 8 weeks of in vivo culture and histologically analyzed. RESULTS: Only implants coated by hydrogel plus cells generated healthy new cartilage. With 3 polymers (high-density polyethylene, acrylic, and extrapurified Silastic), the coverage was nearly complete by elastic cartilage, with minimal fibrocartilage and minimal to no inflammatory reaction. The Food and Drug Administration-approved conventional Silastic implants resulted in fragments of fibrous tissue mixed with elastic cartilage plus evidence of chronic inflammation. The polymethylmethacrylate implant was intermediate in the amount of cartilage formed and degree of inflammation. CONCLUSIONS: This pilot technique combining tissue-engineered autologous elastic cartilage with a permanent biocompatible endoskeleton demonstrated success in limiting the inflammatory response to the scaffold, especially to high-density polyethylene, acrylic, and extrapurified Silastic. This model facilitates the potential to generate tissue of intricate shape, such as the human ear, by internal support. Arch Otolaryngol Head Neck Surg. 2000;126:1448-1452

Engineering autogenous cartilage in the shape of a helix using an injectable hydrogel scaffold.

OBJECTIVE: Previous successful efforts to tissue engineer cartilage for an auricle have used an immunocompromised nude mouse xenograft model. Subsequent efforts in an immunocompetent autogenous animal model have been less successful because of an inflammatory response directed against the foreign scaffold polymer used to provide an auricular shape. We studied an alternative polymer material and surgical technique to engineer autogenous cartilage in the shape of a human ear helix using injectable hydrogel scaffolding, Pluronic F-127 (polyethylene oxide and polypropylene oxide). SUBJECT: Yorkshire swine. MATERIAL AND METHODS: Fresh autogenous chondrocytes were suspended in a biodegradable, biocompatible co-polymer hydrogel, Pluronic F-127, at a concentration of 3 x 10(7) cells/mL. To support the contour of the implant, a skin fold channel in the shape of the helix of a human ear was created in the skin in three sites on the ventral surface of the animal. The cell-hydrogel suspension was injected through the skin fold channel. For controls, injections were made into identical channels using either cells alone or the Pluronic F-127 without cells. After 10 weeks, the specimens were excised and examined both grossly and histologically. RESULTS: Grossly, all implants retained a helical-like shape. Excised specimens possessed flexible characteristics consistent with elastic cartilage. The specimens could be folded and twisted and on release of mechanical pressure would instantly return to the original shape. Histological evaluation of the implants using H&E, Safranin O, trichrome blue, and Verhoeff's stains demonstrated findings consistent with mature elastic cartilage. Control injection of hydrogel alone demonstrated no evidence of cartilage formation and control injection of chondrocytes alone showed evidence only of disassociated elastic cartilage. CONCLUSION: Injection of autologous porcine auricular chondrocytes suspended in a biodegradable, biocompatible hydrogel of Pluronic F-127 resulted in the formation of cartilage tissue in the approximate size and shape of a human ear helix. This preliminary method extends the concept of auricular tissue engineering from an immunocompromised xenograft animal model to an immunocompetent autologous animal model.

Influence of growth factors on tissue-engineered pediatric elastic cartilage.

OBJECTIVE: To investigate the influence of growth factors on tissue-engineered pediatric human elastic cartilage relative to potential clinical application. DESIGN: Controlled study. SUBJECTS: Eleven children ranging in age from 5 to 15 years provided auricular elastic cartilage specimens measuring approximately 1 x 1 x 0.2 cm and weighing approximately 100 mg. INTERVENTIONS: Three million chondrocytes were plated into 4 groups of Ham F-12 culture medium: group 1, Ham F-12 culture medium only; no growth factors (control group); group 2, Ham F-12 culture medium and basic fibroblast growth factor; group 3, Ham F-12 culture medium and transforming growth actor beta; and group 4, Ham F-12 culture medium and a combination of both growth factors. At 3 weeks, the cells were harvested and mixed with a copolymer gel of polyethylene glycol and polypropylene oxide (Pluronic F-127). The cell solution was injected subcutaneously into athymic mice. The constructs were harvested at up to 22 weeks of in vivo culture and histologically analyzed. RESULTS: The average number of cells generated in vitro was as follows: group 1, 12 million; group 2, 40 million; group 3, 7 million; and group 4, 35 million. Group 2 in vivo gross specimens were the largest and heaviest. Histologically, the control group and the basic fibroblast growth factor group (groups 1 and 2) exhibited characteristics compatible with normal auricular cartilage; groups 3 and 4 demonstrated cellular disorganization and moderate to severe fibrous tissue infiltration. CONCLUSIONS: Basic fibroblast growth factor demonstrates the greatest positive influence on the in vitro and in vivo growth of engineered pediatric human auricular cartilage. The results suggest that basic fibroblast growth factor has the potential for clinical application in which a goal will be to generate a large volume of tissue-engineered cartilage from a small donor specimen in a short period of time and of a quality similar to native human elastic cartilage.

Identifying a site for maximum delivery of oxygen to transplanted cells.

For in vivo cell implantation techniques to be successful, the energy and metabolic substrate requirement of the cells being grown must be met. Certain cells with high-energy requirements (e.g., hepatocytes, pancreatic island cells) experience a high degree of cell death after implantation due to a limited supply of oxygen. We proposed that the pleural cavity might be an oxygen-rich environment and hence an excellent site for cell implantation. To test the hypothesis that the delivery of oxygen to the pleural cavity is directly proportional to the inspired oxygen concentration we measured the pO(2) of saline instilled in the pleural cavity as compared to that of the peritoneal cavity. We postulated that the physiologic basis for any difference was the result of direct diffusion of oxygen into the pleural space across the alveoli. The study was conducted on sheep (n = 6), after induction of general anesthesia, in two phases, control and experimental. Saline was instilled into the peritoneal and pleural cavities via catheters, after equilibration at given FiO(2), the pO(2) of the paline aspirated from the two cavities was compared. In the experimental group, animals were sacrificed (no circulation) and ventilated. The same sequence of steps as in the control phase were repeated. In the control group, the pO(2) of saline aspirated from the pleural cavity approached the arterial pO(2) at all FiO(2) levels. The pO(2) of the peritoneal saline aspirate fell over time. In the experimental phase (no circulation), the pO(2) of the pleural cavity saline rose to >400 mm Hg. We conclude that this is a result of direct diffusion and is a potential source of unlimited oxygen supply not dependent on vascular supply.

The science of tissue engineering.

This article reviews the development of tissue engineering during the last decade. The science began to fully develop in association with efforts to combine viable cells with biocompatible material. The history and scope of this new field are presented. Basic principles of cell biology, materials, and technologies are discussed. Future challenges in the field are presented.

An overview of tissue engineered bone.

Numerous important developments in tissue engineering of new bone during the last 10 years are reviewed. Early efforts to combine cells with biocompatible materials are described and applications of this technology are presented with particular focus on uses in orthopaedics and maxillofacial surgery. Basic principles of tissue engineering focusing on cell biology and materials science as used currently in the field are presented. Finally, future challenges are outlined from the perspective of integrating technologies from medicine, biology, and engineering in hopes of translating tissue engineering to clinical applications.