B. Schloo

Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation.

A new approach for tissue creation using synthetic biocompatible and biodegradable polymers as templates onto which cells are seeded is presented. This report concerns the generation of homogeneous plates of stable mature cartilage in vivo. The delivery of chondrocytes on synthetic polymers configured to provide a large surface area for cell attachment and thus to allow cell function and survival by diffusion of nutrients has resulted in the creation of macroscopic plates of up to 100 mg of new cartilage subcutaneously in 19 of 21 animals. The approximate dimensions and configuration of the original templates were maintained as new cartilage was formed and the polymers resorbed.

Long-term engraftment of hepatocytes transplanted on biodegradable polymer sponges.

Hepatocyte transplantation may provide an alternative to orthotopic liver transplantation to treat liver failure. However, suitable systems to transplant hepatocytes and promote long-term engraftment must be developed. In this study, highly porous, biodegradable sponges were fabricated from poly (L-lactic acid) (PLA), and poly (DL-lacticco-glycolic acid) (PLGA), and utilized to transplant hepatocytes into the mesentery of three groups of Lewis rats. The portal vein was shunted to the inferior vena cava in one group of rats (PCS). The second group of animals received a PCS and a 70% hepatectomy on the day of sponge-hepatocyte implantation (PCS + HEP), and the control group (CON) received no surgical stimulation. The sponges were vascularized by ingrowth of fibrovascular tissue over the first 7 days in vivo. Approximately 95-99% of the implanted hepatocytes (determined utilizing computer-assisted image analysis) died in all three experimental groups during this time. The number of engrafted hepatocytes in the CON group further decreased over the next 7 days to 1.3 +/- 1.1% of the original cell number. However, the number of engrafted hepatocytes in the PCS and PCS + HEP increased over this time to 6 +/- 1% and 5 +/- 2%, respectively. The number of engrafted hepatocytes in the PCS group continued to increase over the next 2.5 months to a value of 26 +/- 12% of the initial cell number, and a large number of engrafted hepatocytes was still present at 6 months. These results indicate that stable new tissues can be engineered by transplanting hepatocytes on biodegradable sponges into heterotopic locations if appropriate stimulation is provided.

De novo cartilage generation using calcium alginate-chondrocyte constructs.

&NA; These studies investigated the utility of calcium alginate as a biocompatible polymer matrix within which large numbers of chondrocytes could be held successfully in a three‐dimensional structure and implanted. Further, the ability of chondrocyte‐calcium alginate constructs to engraft and generate new cartilage was examined. Chondrocytes isolated from calf shoulders were mixed with a 1.5% sodium alginate solution to generate cell suspensions with densities of 0, 1.0, 5.0, and 10.0 × 106 chondrocytes/ml. The cell suspensions were gelled to create disks that were placed in subcutaneous pockets on the dorsums of nude mice. The alginate concentration and CaCl2 concentration used to make the disks also were varied. A total of 20 mice were implanted with 67 bovine chondrocyte‐calcium alginate constructs. Samples with an initial cellular density of at least 5.0 × 106 chondrocytes/ml demonstrated gross cartilage formation 12 weeks alter implantation. Cartilage formation was observed microscopically in specimens with a cellular density as low as 1.0 × 106 chondrocytes/ ml. The histoarchitecture of the new cartilage closely resembled that of native cartilage. Cartilage formation was independent of CaCl2 concentration (15 to 100 mM) or alginate concentration (0.5% to 4.0%) used in gel polymerization. (Plast. Reamslr. Surg. 97: 168, 1996.)

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.

Pulmonary growth and remodeling in infants with high-risk congenital diaphragmatic hernia

Infants born with congenital diaphragmatic hernia (CDH) have pulmonary hypoplasia, but the pattern of postnatal growth in these lungs has not been documented. The lungs of 21 children dying with CDH were analyzed to determine how the pulmonary morphology changed with age. The patients were stratified into three age groups for ANOVA analysis (less than 8 days, 8 to 21 days, greater than 21 days). Morphometric techniques previously described were used. Lung volume and weight as well as pulmonary artery length and diameter increased with age (P = .04), whereas the number of airway generations was similar for each group. Radial alveolar number also increased, particularly in the contralateral lung (P = .02). The percentage of intraacinar artery muscularization decreased with age (P = .02), while larger intraacinar arteries showed a nonmuscular structure, again particularly in the contralateral lung (P = .004). It is concluded that: (1) significant lung growth does occur postnatally at the alveolar level after CDH repair; and (2) there is postnatal vascular remodelling resulting in larger and less muscular arteries. These changes should contribute to a decrease in pulmonary arterial hypertension over time. However, the time period over which these changes occur exceeds the current limitations of invasive support measures such as extracorporeal membrane oxygenation. Elucidation of the factors responsible for this growth could result in new therapeutic strategies to enhance or accelerate postnatal pulmonary development in infants with CDH.

Femoral shaft reconstruction using tissue-engineered growth of bone

Tissue engineering is an interdisciplinary field that applies the principles and methods of engineering and the life sciences to the development of biologic substitutes. Bovine periosteum-derived cells were cultivated in vitro, put onto bioresorbable polymer fiber constructs, and allowed to grow until most of the fibers were coated with multiple layers of osteoblasts. Standardized 9-mm nonhealing defects were created in 24 male athymic rats femurs and bridged with titanium miniplates. In 12 animals, the defects were filled with polymer constructs containing periosteum-derived cells (experimental group); in another 12 animals, the defects were either left unfilled (control group I) or filled with polymer templates alone (control group II). After 12-week in vivo implantation, the new bone produced bridged the surgically created defects completely in seven of 10 cases. The animals of the control groups did not show significant bone formation in the gap. Histologic evaluation revealed bone formation in all experimental specimens with rests of cartilage islands showing hypertrophying chondrocytes indicative of enchondral bone formation. Tissue-engineered growth of bone resulted in healing of large segmental bone defects in an orthotopic site in an animal model. The findings of this study support potential applications of the technique of tissue-engineered growth of bone to clinical situations where local bone formation is needed.

Localized delivery of epidermal growth factor improves the survival of transplanted hepatocytes

Hepatocyte transplantation may provide a new approach for treating a variety of liver diseases if a sufficient number of the transplanted cells survive over an extended time period. In this report, we describe a technique to deliver growth factors to transplanted hepatocytes to improve their engraftment. Epidermal growth factor (EGF) was incorporated (0.11%) into microspheres (19 ± 12 μm) fabricated from a copolymer of lactic and glycolic acid using a double emulsion technique. The incorporated EGF was steadily released over 1 month in vitro, and it remained biologically active, as determined by its ability to stimulate DNA synthesis, cell division, and long‐term survival of cultured hepatocytes. EGF‐containing microspheres were mixed with a suspension of hepatocytes, seeded onto porous sponges, and implanted into the mesentery of two groups of Lewis rats. The first group of animals had their portal vein shunted to the inferior vena cava prior to cell transplantation (portal‐caval shunt = PCS), and the second group of animals did not (non‐PCS). This surgical procedure improves the survival of transplanted hepatocytes. The engraftment of transplanted hepatocytes in PCS animals was increased two‐fold by adding EGF microspheres, as compared to adding control microspheres that contained no growth factors. Devices implanted into non‐PCS animals had fewer engrafted hepatocytes than devices implanted into PCS animals, regardless of whether blank or EGF‐containing microspheres were added. These results first indicate that it is possible to design systems which can alter the microenvironment of transplanted hepatocytes to improve their engraftment. They also suggest that hepatocyte engraftment is not improved by providing single growth factors unless the correct environment (PCS) is provided for the transplanted cells.

Studies in rat liver perfusion for optimal harvest of hepatocytes

Pediatric liver transplantation is successful but donor scarcity is a major limitation. We are studying hepatocyte transplantation as an alternative to provide functional hepatic replacement. This report details the study of rat liver perfusion for optimal harvest of hepatocytes and cell implantation. We performed 128 rat liver perfusions using a technique modified from the two-step enzymatic perfusion described by Seglen. We examined variations in the perfusion, rate, time, antegrade versus retrograde, pulsatile versus continuous flow, temperature, collagenase type, and variables of buffer composition. We have found optimal cell yield and viability under the following conditions: in situ perfusion, continuous flow at 25 cc/min, retrograde perfusion via the inferior vena cava, water bath temperature 38 degrees C, Boerhinger-Mannheim collagenase using a nonoxygenated HEPES based perfusion buffer, pH 7.4, for the initial perfusion and the same buffer with 4.8 mmol/L CaCl2 for the collagenase perfusion. These conditions consistently generate cell harvests of 500 to 700 x 10(5) cells/g of liver tissue with cell viability between 85% and 95%.

The mesentery as a laminated vascular bed for hepatocyte transplantation.

The small bowel mesentery provides a unique structure of a large vascularized surface area to support hepatocyte transplantation. Cell-seeded polymeric matrices can be juxtaposed in a relatively atraumatic manner between leaves of mesentery such that adequate exchange of nutrients and diffusion of gases can proceed in the interim while neovascularization occurs. Hepatocytes obtained from (RHA) Wistar rats by collagenase perfusion were seeded onto non-woven filamentous sheets of polyglycolic acid 1 × 3 cm in size and 2 mm thickness to a density of 500,000 cells/cm2. Twenty-six recipient Gunn rats (UDP-glucuronyl transferase deficient) underwent laparotomy. Hepatocyte-ladened polymer sheets were placed between leaves of mesentery. Eight sheets were placed per animal and the leaves were approximated, creating a functional implant 1 × 3 × 2 cm. Biopsies between 5-99 days after implantation revealed neovascularization, moderate inflammatory reaction and the presence of viable hepatocytes in 96% (25/26). Immunoperoxidase studies using anti-albumin antibody substantiated hepatocyte specific function in implants. HPLC profiles of bile from Gunn rats transplanted with hepatocytes from congeneic (RHA) rats demonstrated the presence of bilirubin conjugates. There were no conjugation fractions seen in control gunn rats without hepatocyte transplantation. Although total serum bilirubin did not significantly decrease, conjugated bilirubin was identified in 46% (12/26) animals after transplantation with congeneic hepatocytes. We conclude that the mesentery of the small bowel provides a large vascularized surface for cell transplantation. Large numbers of metabolically active hepatocytes can engraft, vascularize, and show function. The mesentery may be a potential bed for clinical hepatocyte transplantation.

Hepatocyte Transplantation in Swine Using Prevascularized Polyvinyl Alcohol Sponges

Hepatocyte transplantation shows promise as therapy to support liver function. We have shown that hepatocytes can be transplanted into prevascularized synthetic polymers in rat models. While there are many studies in rats showing the benefits of hepatocyte transplantation, there are few in larger animal models more akin to humans. Therefore, we developed a swine model of hepatocyte transplantation using prevascularized synthetic polymers. Polyvinyl alcohol sponges measuring 2 x 3 x 0.5 cm were implanted in preperitoneal, mesenteric, and subcutaneous spaces for prevascularization as a transplantation bed. On postimplantation day 0, 4, 8, or 12 the sponges were removed and examined histologically. New tissue ingrowth was quite satisfactory on day 8 and 12 in preperitoneal and mesenteric sites, but the sponges in subcutaneous tissue were compressed, leaving little space for hepatocyte engraftment. Mesenteric sponges were irritating to intraabdominal organs and induced peritoneal adhesions. Thus, the preperitoneal sponges seemed to be best for hepatocyte transplantation. Hepatocytes isolated from donor livers using collagenase perfusion were transplanted into the preperitoneal sponges prevascularized for 0, 4, 8, or 12 days as allografts with cyclosporine immunosuppression, and the number of hepatocytes was evaluated on posttransplantation day 4. The optimal period for prevascularization for subsequent hepatocyte implantation was 8 days. The number of hepatocytes implanted in the preperitoneal sponges prevascularized for 8 days was counted on day 0, 1, 4, or 8 after transplantation. Hepatocytes were lost mainly in the first day after implantation, but still many hepatocytes maintained their shape histologically and there were mitotic figures confirming growth of the transplanted hepatocytes. Areas of hepatocytes within the sponge devices showed tissue remodeling with plates of hepatocytes lined with sinusoid-like capillaries and evidence of early tubular formation. Positive staining by immunohistochemical examination using antipig albumin indicated albumin production by implanted hepatocytes. The effect of a portacaval shunt as a hepatotrophic stimulation to maintenance of the implanted hepatocytes was evaluated on day 4, 8, or 12 after implantation. Total hepatocyte number in sponges was significantly increased by portacaval shunt compared to controls treated by a sham operation. These results suggest that significant numbers of hepatocytes can engraft and function using a prevascularized polymer bed as a site for transplantation and ongoing hepatotrophic stimulation with portacaval shunting.