Joseph Upton

Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear

&NA; This study evaluates the feasibility of growing tissue‐engineered cartilage in the shape of a human ear using chondrocytes seeded onto a synthetic biodegradable polymer fashioned in the shape of a 3‐year‐old childs auricle. A polymer template was formed in the shape of a human auricle using a nonwoven mesh of polyglycolic acid molded after being immersed in a 1% solution of polylactic acid. Each polyglycolic acid‐polylactic acid template was seeded with chondrocytes isolated from bovine articular cartilage and then implanted into subcutaneous pockets on the dorsa of 10 athymic mice. The three‐dimensional structure was well maintained after removal of an external stent that had been applied for 4 weeks. Specimens harvested 12 weeks after implantation and subjected to gross morphologic and histologic analysis demonstrated new cartilage formation. The overall geometry of the experimental specimens closely resembled the complex structure of the childs auricle. These findings demonstrate that polyglycolic acid‐polylactic acid constructs can be fabricated in a very intricate configuration and seeded with chondrocytes to generate new cartilage that would be useful in plastic and reconstructive surgery. (Plast. Reconstr. Surg. 100: 297, 1997.)

Altered Growth and Branching Patterns in Synpolydactyly Caused by Mutations in HOXD13

Hox genes regulate patterning during limb development. It is believed that they function in the determination of the timing and extent of local growth rates. Here, it is demonstrated that synpolydactyly, an inherited human abnormality of the hands and feet, is caused by expansions of a polyalanine stretch in the amino-terminal region of HOXD13. The homozygous phenotype includes the transformation of metacarpal and metatarsal bones to short carpal- and tarsal-like bones. The mutations identify the polyalanine stretch outside of the DNA binding domain of HOXD13 as a region necessary for proper protein function.

A Mosaic Activating Mutation in AKT1 Associated with the Proteus Syndrome

BACKGROUND The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state. METHODS We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots. RESULTS Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation. CONCLUSIONS The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K-AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.).

TRANSPLANTATION OF CELLS IN MATRICES FOR TISSUE REGENERATION

Tissue engineering is a field that has truly emerged in the last decade. It has brought together diverse technologies, e.g. cell culture, polymer chemistry and transplantation. The creation of matrices to guide tissue regeneration allows manipulation at several levels, i.e. the cells employed, the choice of polymer and the design of construct assembly methods. We present experience using such constructs to guide regeneration of diverse tissues, e.g. liver, intestine, urologic tissue, skin, cartilage, bone and cardiovascular structures. Emerging concepts in using cell/polymer constructs include the need for appropriate modeling of the micromechanical environments of different tissues, as well as the necessity of finding new strategies to achieve vascularization of tissues for transplant. Finally, the concept of applying tissue-engineered structures to non-native sites is discussed.

Experience with the temporoparietal fascial free flap

The temporoparietal fascia is an ideal tissue source for free transfer to distant sites where ultrathin coverage is either desirable or mandatory. The fascias dependable vascular anatomy facilitates the technical aspects of microvascular transfer by means of its large vessels, ample pedicle, and ability to be grafted on either side. Furthermore, this highly vascular tissue is available in surprisingly large quantities, and its donor scar is hidden in the hair. The authors have found this flap useful (1) in covering exposed bone and tendon without adding unwanted bulk, (2) in providing thin flap coverage or lining in major facial reconstruction, (3) in covering vital structures such as exposed nerves and vessels, (4) in providing neovascularity both as a recipient graft bed and for control of chronic infection, and (5) in reestablishing gliding-tendon mechanisms. The authors have successfully employed this free flap in 15 cases which involved deformities of the ankle, foot, Achilles tendon, forearm, hand, nose, and contralateral ear and scalp. Seven cases are utilized to illustrate the broad application of this unique and versatile free flap.

Cartilage engineered in predetermined shapes employing cell transplantation on synthetic biodegradable polymers.

Cartilage is often used as structural support tissue for cosmetic repair in plastic and reconstructive surgery. We describe the efficacy of a new approach for the generation of cartilage in predetermined shapes using specially configured biodegradable synthetic polymer devices as delivery vehicles for transplanted cells. Synthetic biodegradable polymer scaffolds were configured in one of four specific shapes, i.e., a triangle, a rectangle, a cross, and a cylinder. The polymer matrices were seeded with freshly isolated bovine articular chondrocytes and then implanted subcutaneously into nude mice. Gross examination of excised specimens 12 weeks after implantation revealed the presence of new hyaline cartilage of approximately the same dimensions as the original construct. This cartilage showed no signs of resorption or overgrowth over the 12-week time course of the experiment. Histologie evaluation using hematoxylin and eosin stains confirmed the presence of normal mature hyaline cartilage in 46 of 48 specimens. These results suggest that cartilage can be created in predetermined shapes and dimensions using cell transplantation on appropriate polymer templates. This technology would be useful in cosmetic and reconstructive surgery.

Skeletal changes associated with vascular malformations.

Five hundred and eighty birthmarks were reviewed; 356 were hemangiomas and 224 were malformations. Bony alterations occurred in association with only 1 percent of hemangiomas, in contrast with 34 percent of patients with vascular malformations. These alterations in bone development were classified according to size, shape, and density changes. Hypertrophy and distortion were typical of lymphatic malformations. Hypoplasia and demineralization were characteristic findings in the extremity venous malformations. Destructive and intraosseous changes were more commonly noted in the arterial or high-flow lesions. Possible mechanisms of altered skeletal growth include mechanical, physiological, and developmental processes.

Design of nasoseptal cartilage replacements synthesized from biodegradable polymers and chondrocytes.

Reconstructive and aesthetic surgery of the nose is a challenging problem in facial plastic surgery. In this study, biodegradable polymers composed of polyglycolic acid (PGA) and poly-L-lactic acid (PLLA) and their co-polymers were used to produce templates to transplant cells and promote regeneration of structural cartilage. A highly porous anatomically shaped three-dimensional non-woven PGA fibre network was sprayed with a coating polymer solution. Reinforcement of the outer circumference of the 12 nasoseptal constructs using high molecular weight PLLA further stabilized the constructs during the process of neomorphogenesis of cartilage, both during in vitro incubation and in vivo implantation. These cell transplantation devices also proved to be adhesive substrates for dissociated bovine chondrocytes. When implanted subcutaneously into nude mice, the polymer templates guided the reorganization after 8 wk of the bovine chondrocytes into neocartilage in the precisely designed size and shape of the original size and shape of the polymer delivery device. All implants loaded with chondrocytes showed evidence of formation of histologically organized hyaline cartilage. The implantation of nasal scaffolds without cells did not show cartilage formation. The technique of tissue engineered growth of cartilage has potential applications in orthopaedic, plastic and reconstructive, and craniomaxillofacial surgery.

Formation of phalanges and small joints by tissue-engineering.

BACKGROUND This report describes the formation of small phalanges and whole joints from three types of bovine-cell sources transplanted onto biodegradable polymer matrices. The resulting structures had the shape and composition of human phalanges with joints. METHODS Fresh bovine periosteum was wrapped around a copolymer of polyglycolic and poly-L-lactic acid. Separate sheets of polyglycolic acid polymer were then seeded with chondrocytes and tenocytes isolated from the shoulders of freshly killed calves. The gross form of a composite tissue structure was constituted in vitro by assembling the parts and suturing them to create models of a distal phalanx, a middle phalanx, and a distal interphalangeal joint. RESULTS Subcutaneous implantation of the sutured composite tissues into athymic mice resulted in the formation, after twenty weeks, of new tissue with the shape and dimensions of human phalanges with joints. Histological examination revealed mature articular cartilage and subchondral bone with a tenocapsule that had a structure similar to that of human phalanges and joints. There was continuous cell differentiation at the ectopic site even after extended periods. CONCLUSIONS These findings suggest that the formation of phalanges and small joints is possible with the selective placement of periosteum, chondrocytes, and tenocytes into a biodegradable synthetic polymer scaffold.

Experimental tracheal replacement using tissue-engineered cartilage

The authors tested the feasibility of using tissue-engineered cartilage, grown in the shape of cylinders, for replacing large circumferential defects of the cervical trachea in rats. Chondrocytes obtained from the shoulder of newborn calves were seeded onto a synthetic nonwoven mesh, 100 microns thick, of polyglycolic acid fibers 15 microns in diameter, cut into pieces of 2.5 x 4 cm. Twenty cell-polymer constructs were wrapped around silastic tubes and implanted into 10 nude mice for 4 weeks. Specimens were then excised and evaluated grossly and histologically for the presence of new cartilage, and biomechanically for their ability to resist collapse upon application of negative pressure. Six cylinders of tissue-engineered cartilage were then sutured into large circumferential defects created in the cervical tracheas of nude rats to replace the excised trachea. Implantation of cell-polymer constructs resulted in the formation of cylinders of hyaline cartilage. When placed within the lumen of a segment of bowel denuded of its mucosal lining, the hollow cylinders resisted collapse in all instances upon administration of negative 200 mm Hg pressure. The cartilage was grossly and histologically identical to that from which the cells had been initially isolated. Four of the six animals receiving these cartilage cylinders as tracheal replacements survived the procedure and were able to breathe in an unassisted fashion. Three of these animals never recovered fully from the anesthetic and the operation, and expired at 24, 48, and 72 hours. The fourth animal fully recovered from the procedure, and breathed spontaneously for 1 week, with no apparent limitations. Increasing respiratory distress then developed, and the animal died.(ABSTRACT TRUNCATED AT 250 WORDS)