David P. Mooney

Tissue Engineering by Cell Transplantation Using Degradable Polymer Substrates

This paper reviews our research in developing novel matrices for cell transplantation using bioresorbable polymers. We focus on applications to liver and cartilage as paradigms for regeneration of metabolic and structural tissue, but review the approach in the context of cell transplantation as a whole. Important engineering issues in the design of successful devices are the surface chemistry and surface microstructure, which influence the ability of the cells to attach, grow, and function normally; the porosity and macroscopic dimensions, which affect the transport of nutrients to the implanted cells; the shape, which may be necessary for proper function in tissues like cartilage; and the choice of implantation site, which may be dictated by the total mass of the implant and which may influence the dimensions of the device by the available vascularity. Studies show that both liver and cartilage cells can be transplanted in small animals using this approach.

Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis.

Publisher Summary This chapter focuses on the role of the intracellular cytoskeleton (CSK) in cell shape determination and tissue morphogenesis. The role of mechanical changes in the CSK during embryological development is reviewed. The chapter focuses on the mechanism by which mechanical forces are transmitted across the cell surface and through the CSK, as well as how they regulate cell shape. An analysis of the biomechanical basis of cell shape control addresses two central questions: (1) how do changes in mechanical forces alter CSK organization, and (2) how do changes in CSK structure regulate cell growth and function. The results from recent studies showing that the CSK can respond directly to mechanical stress are also reviewed. The particular type of mechanical response that living cells exhibit is consistent with a theory of CSK architecture that is based on tensional integrity and is known as “tensegrity”. Inherent to the tensegrity model is an efficient mechanism for integrating changes in structure and function at the tissue, cell, nuclear, and molecular levels. The chapter explores the possibility that CSK tensegrity may also provide a mechanical basis for cell locomotion as well as a structural mechanism for coupling mechanical and chemical signaling pathways inside the cell.

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.

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.

Tissue-engineered growth of cartilage: the effect of varying the concentration of chondrocytes seeded onto synthetic polymer matrices

Ninety-six synthetic bioresorbable cell-delivery devices (10 x 10 x 0.5 mm) were seeded, varying the concentrations of living chondrocytes (2, 10, 20, 100 million cells/cc) isolated from shoulders of freshly killed calves and implanted subcutaneously on the dorsum of nude mice after 1 week of in vitro culture. This resulted in the formation of new cartilage in 95.6% of the implants. Twenty-four control implants (0 cells seeded) did not show cartilage formation. During 12 weeks of in vivo implantation, the wet weight and the thickness of the specimens (10, 20, 100 million cells/cc) increased significantly. Histologic analysis revealed cells appearing in their own lacunar structures surrounded by basophilic matrix. The increase in sulfated glycosaminoglycan content indicated the maturation of the extracellular matrix. The ability to manipulate the growth of new cartilage on biocompatible polymer scaffolds by varying the cell density before in vivo implantation will allow engineering to optimize the utilization of chondrocytes in relation to the desired shape, thickness, and quality of the new cartilage.

Soft-tissue augmentation with injectable alginate and syngeneic fibroblasts

Tissue engineering, a field that combines polymer scaffolds with isolated cell populations to create new tissue, may be applied to soft-tissue augmentation—an area in which polymers and cell populations have been injected independently. We have developed an inbred rat model in which the subcutaneous injection of a hydrogel, a form of polymer, under vacuum permits direct comparison of different materials in terms of both histologic behavior and their ability to maintain the specific shape and volume of a construct. Using this model, we compared three forms of calcium alginate, a synthetic hydrogel, over an 8-week period—standard alginate that was gelled following injection into animals (alginate post-gel), standard alginate that was gelled before injection into animals (alginate pre-gel) and alginate-RGD, to which the cell adhesion tripeptide RGD was linked covalently (RGD post-gel). Parallel groups that included cultured syngeneic fibroblasts suspended within each of these three gels were also evaluated (alginate post-gel plus cells, alginate pre-gel plus cells, and RGD post-gel plus cells). The study used 54 inbred Lewis rats (n = 9 for each of the six groups). Construct geometry was optimally maintained in the alginate post-gel group in which 58 percent of the original volume was preserved at 8 weeks and increased to 88 percent at 8 weeks when syngeneic fibroblasts were included within the gel. Volume was not as well preserved in the RGD post-gel group (25 percent of original volume at 8 weeks), but again increased when syngeneic fibroblasts were included (41 percent of original volume at 8 weeks). Maintenance of volume was poorest in the alginate pre-gel group (31 percent of original volume at 8 weeks) and failed to be augmented by the addition of fibroblasts (19 percent of original volume at 8 weeks). Histologically, the gel remained a uniform sheet surrounded by a fibrous capsule in the alginate post-gel groups. In the alginate pre-gel and RGD post-gel groups, there was significant ingrowth of a fibrovascular stroma into the gel with fragmentation of the construct. In constructs in which syngeneic fibroblasts were included, cells were visualized throughout the gel but did not extend processes or appear to contribute to new tissue formation. Material compression testing indicated that the alginate and RGD post-gel constructs became stiffer over a 12-week period, particularly in the cell-containing groups. Our results suggest that calcium alginate could be a suitable agent for soft-tissue augmentation when gelled subcutaneously following injection. The addition of syngeneic fibroblasts enhanced the ability of the gel to maintain the volume of a construct; this seems to be mediated by increased gel stiffness rather than by de novo tissue formation. Our animal model, in combination with material testing data, permits rigorous comparison of different materials used for soft-tissue augmentation.

Tumor necrosis factor and wound healing.

Tumor necrosis factor alpha (TNF), 1 to 500 ng in saline (PBS) or collagen, was applied to the wounds of normal and Adriamycin-impaired mice and wound disruption strength (WDS) and histology were examined. Also wounded mice were administered TNF 25 to 75 micrograms/kg IP daily and WDS was determined. Wound histology was examined 6 months after wounding and local TNF application. Local TNF 5 to 500 ng in PBS did not significantly affect WDS. Local TNF 5 to 50 ng in collagen increased WDS 33% to 65% in Adriamycin-impaired animals (p = 0.05 to p less than 0.02). Local TNF 50 to 500 ng in collagen increased WDS 23% to 49% in normal animals (p = 0.08 to p less than 0.01). Adriamycin-impaired animals demonstrated improved wound histology with local TNF in collagen. Systemic TNF did not significantly affect WDS. Local TNF in collagen did not induce histologic pathology at 6 months. TNF may modulate macrophage function and local TNF in collagen can improve WDS in normal and Adriamycin-impaired animals.

A Randomized Clinical Trial of the Management of Esophageal Coins in Children

Context. Children frequently ingest coins. When lodged in the esophagus, the coin may cause complications and must either be removed or observed to pass spontaneously. Objectives. (1) To compare relatively immediate endoscopic removal to a period of observation followed by removal when necessary and (2) to evaluate the relationship between select clinical features and spontaneous passage. Design/Setting. Randomized, prospective study of children <21 years old who presented to an emergency department with esophageal coins in the esophagus. Exclusion criteria were (1) history of tracheal or esophageal surgery, (2) showing symptoms, or (3) swallowing the coin >24 hours earlier. Children were randomized to either endoscopic removal (surgery) or admission for observation, with repeat radiographs ∼16 hours after the initial image. Outcome Measures. Proportion of patients requiring endoscopic removal, length of hospital stay, and the number of complications observed. Results. Among 168 children who presented with esophageal coins lodged in the esophagus, 81 were eligible. Of those eligible, 60 enrolled, 20 refused consent, and 1 was not approached. In the observation group, 23 of 30 (77%) children required endoscopy compared with 21 of 30 (70%) in the surgical group. Total hospital length of stay was longer in the randomized-to-observation group compared with the randomized-to-surgery group (mean: 19.4 [SD: ±8.0] hours vs 10.7 [SD: ±7.1] hours, respectively). There were no complications in either group. Spontaneous passage occurred at similar rates in both groups (23% vs 30%). Spontaneous passage was more likely in older patients (66 vs 46 months) and male patients (odds ratio: 3.7; 95% confidence interval: 0.98–13.99) and more likely to occur when the coin was in the distal one third of the esophagus (56% vs 27% [95% confidence interval: 1.07–5.57]). Conclusions. Because 25% to 30% of esophageal coins in children will pass spontaneously without complications, treatment of these patients may reasonably include a period of observation, in the range of 8 to 16 hours, particularly among older children and those with distally located coins.

Congenital duodenal obstruction: A 32-year review†

Although survival in infants with congenital intestinal obstruction has improved, duodenal obstruction continues to present unique challenges. One hundred thirty-eight newborns and infants (aged 0 to 30 days) were treated for congenital duodenal obstruction. Sixty-five were boys and 73 were girls. Sixty-one (45%) were premature. Forty-six had an intrinsic defect (atresia, web, stenosis, or duplication), 64 had an extrinsic defect (annular pancreas or malrotation with congenital bands), while 28 had various combinations of these. Presenting signs included vomiting (90%, bilious in 66%), abdominal distention (25%), dehydration (24%), and weight loss (17%). Although plain film abdominal x-ray was diagnostic in 58%, upper and/or lower gastrointestinal contrast studies were obtained in 71% of infants to confirm diagnosis. Thirty-eight percent of patients had associated anomalies, including Downs syndrome (11%), cardiac defects, other atresia, other trisomy syndrome, imperforate anus, and central nervous system anomalies. Fourteen patients (10%) had 3 or more other anomalies, many of which required additional surgical therapy. The operative repair of the various defects included Ladds procedure for malrotation (31%), duodenoduodenostomy (14%), duodenojejunostomy (22%), gastrojejunostomy or gastroduodenostomy (4%), excision of the web and duodenoplasty (3%), or combination of the above (22%). Gastrostomy was placed in 61%. One hundred twenty-eight patients survived (93%). The causes of death were combinations of sepsis, pneumonia, brain hemorrhage, short bowel, and cardiac anomaly. Eight of 10 (80%) who died had other serious anomalies. Twenty patients (14%) required reoperation 5 days to 4 years postoperatively for obstructing lesions (5), wound dehiscence (3), anastomotic leak or dysfunction (6), other atresias (2), choledochal cyst (1), pyloric stenosis (1), and gastroesophageal reflux (2).(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of brush border enzymes, basement membrane components, and electrophysiology of tissue-engineered neointestine

BACKGROUND/PURPOSE Previous studies have shown that intestinal crypt cell transplantation using biodegradable scaffolds can generate stratified epithelium reminiscent of embryonic gut. The authors propose to tissue engineer small intestine on biodegradable scaffolds by transplanting intestinal epithelial organoid units, which maintain the epithelial mesenchymal cell-cell interaction necessary for epithelial survival, proliferation, and differentiation. METHODS Intestinal epithelial organoid units were isolated from neonatal Lewis rats by enzyme digestion and differential sedimentation. Organoid units were seeded on to tubular scaffolds made of nonwoven polyglycolic acid (PGA) sprayed with 5% polylactic acid (PLA). Polymers either were coated (28 constructs) or noncoated (33 constructs) with collagen type I. A total of 61 organoid unit polymer constructs were implanted into 61 animals. Animals were killed and constructs harvested at 2, 6, 7, 8, 9, 10, 12, and 14 weeks. RESULTS Histological analysis showed formation of neomucosa characterized by columnar epithelium with goblet, and paneth cells were evident in 47 of the 61 constructs. The outer walls were composed of fibrovascular tissue, degradable polymer, extracellular matrix, and smooth muscle-like cells. Immunofluorescent microscopy showed apical staining of brush border enzymes, sucrase and lactase, and basolateral staining for laminin, indicating the establishment of cell polarity. Electrophysiology of Ussing-chambered neomucosa and adult ileal mucosa exhibited similar transepithelial resistance. CONCLUSION These results suggest that intestinal crypt cells heterotopically transplanted as epithelial organoid units on PGA-PLA tubular scaffolds can survive, reorganize, and regenerate complex composite tissue resembling small intestine demonstrating organ morphogenesis, cytodifferentiation, and phenotypic maturation.