Evaluation of methods of hepatotrophic stimulation in rat heterotopic hepatocyte transplantation using polymers.

BACKGROUND: Hepatocyte transplantation has been studied as an alternative to organ transplantation. Hepatocyte transplant models should provide sufficient cell mass for replacement function and hepatotrophic stimulation of the transplanted cells in heterotopic locations. METHOD: The authors used three-dimensional porous polyvinyl-alcohol matrices as cell carriers, which were implanted between mesenteric leaves of the intestine. In this study, different methods were evaluated for hepatotrophic stimulation. Fifty million transplanted hepatocytes (approximately 10% liver mass) were implanted in Lewis rats. We compared 70% partial hepatectomy, portacaval shunt, cotransplantation of enterocytes, cotransplantation of islets of Langerhans, and methylprednisolone injection to a control group with only hepatocyte transplantation. Portacaval shunt and islet cotransplantation also were used in combination. Specimens were harvested 2 weeks after transplantation, and area per histological cross section compromised by hepatocytes was measured. RESULTS: Seventy percent partial hepatectomy, enterocyte cotransplantation, and methylprednisolone injection resulted in hepatocyte maintenance similar to control group (3,100 +/- 7,592 microm2). Portacaval shunt (96,866 +/- 55,039 microm2) and islet cotransplantation (173,020 +/- 75,977 microm2) yielded a highly significant increase in hepatocyte area. The combination of portacaval shunt and islet cotransplantation resulted in a significant increase compared with using these methods individually (288,930 +/- 86,726 microm2). Additional immunohistochemical stains for active DNA synthesis, insulin, and glucagon demonstrated the proliferative abilities of the hepatocytes and the synthesis of insulin and glucagon in the cotransplanted islets. CONCLUSION: Hepatocyte transplantation can be performed using polymer carriers and that hepatocyte survival and maintenance can be improved with portacaval shunt and islet cotransplantation.

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.

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 mum) 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. (c) 1996 John Wiley & Sons, Inc.

Chronic pulmonary hypertension in sheep: temporal progression of lesions.

Scant data exist on the evolution of the lesions of pulmonary hypertension. This study establishes a model in sheep in which the left upper lobe (LUL) was rendered hypertensive by a systemic-pulmonary shunt while the rest of the pulmonary circulation remained normotensive. By examining lung tissue at 2 months and 1 1/2 years after shunting, we sought the temporal progression of pulmonary hypertensive lesions. In the hypertensive LULs (n = 5), many vascular lesions were seen in contrast to the absence of lesions in both the contralateral normotensive lungs (n = 5) and the "control" lungs from sheep which underwent thoracotomy without shunting (n = 5). Vascular necrosis and vasculitis were present after 2 months (P < 0.01) but disappeared after 1 1/2 years. In contrast, intimal thickening was present after 1 1/2 years (n = 2, P < 0.01) but not significantly after 2 months. These intimal lesions often demonstrated increased cellularity staining positively for factor VIII. Plexiform lesions were present at 2 months (P < 0.05) but were more profuse after 1 1/2 years (P < 0.01). These findings are consistent with an early vascular injury and a later remodeling or reparative process in hemodynamic pulmonary hypertension.

De novo cartilage generation using calcium alginate-chondrocyte constructs.

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 x 10(6) 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 x 10(6) chondrocytes/ml demonstrated gross cartilage formation 12 weeks after implantation. Cartilage formation was observed microscopically in specimens with a cellular density as low as 1.0 x 10(6) 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.

Development of biomechanical properties and morphogenesis of in vitro tissue engineered cartilage.

Neocartilage was engineered by culturing bovine chondrocytes on poly(glycolic acid) (PGA) fibrous nonwoven scaffolds. The biomechanical properties and morphologies of the PGA-chondrocyte constructs were studied over 12 weeks of in vitro culture. PGA scaffolds without cells lost their mechanical strength and structural integrity between week 2 and week 3 in culture. The thickness of the PGA-chondrocyte constructs decreased by 35% during the first 3 weeks, but the thickness increased from week 3 to week 9 to a thickness 42% higher than that of the starting scaffolds, which was then maintained. Safranin O staining of PGA-chondrocyte constructs revealed increasing proteoglycan formation over time. The compressive modules of PGA-chondrocyte constructs increased with in vitro culture time, and reached the same order of magnitude as that of normal bovine cartilage at week 9. The aggregate modulus of the PGA-chondrocyte constructs decreased by 57% over the first 2 weeks but then increased, reaching the same order of magnitude as normal bovine cartilage at week 12. The apparent permeability of the PGA-chondrocyte constructs, which was initially four orders of magnitude above that of normal cartilage, decreased between weeks 1 and 3 and thereafter remained the same order of magnitude as that measured for normal cartilage.

Human hepatocyte isolation and transplantation into an athymic rat, using prevascularized cell polymer constructs.

Human hepatocyte viability and function in vivo in an athymic rat was assessed after transplantation on prevascularized polymer constructs with hepatotrophic stimulation. Sixteen liver biopsy specimens, weighing 5 to 12 g, were obtained from the New England Organ Bank and from the operating room after liver resection. In the laboratory they were catheterized and perfused to obtain liver cell suspensions. From eight of the 16 cell suspensions, only in vitro studies were performed. They showed 40% cell attachment 24 hours after initial cell plating. For patients aged 2, 35, and 60 years, they showed a 20% increase, a 1% decrease, and a 57% decrease (respectively) in cell number from day 2 to day 4, after cell plating. Eight cell suspensions were transplanted into athymic rats. On sections examined histologically, implanted hepatocytes were seen within the fibroblast ingrowth, in the space of the polymer device, until day 21 after cell injection. On day 9 after hepatocyte injection, reorganized hepatic parenchyma was seen on the tissue section. Implanted hepatocyte areas, quantitated through morphometric analysis on days 0, 3, and 7, showed a 36% increase in engraftment 3 days after injection, and a 42% decrease 7 days after injection. At the same time-points, immunoperoxidase staining visualized intracellular albumin, which was specific for the implanted hepatocytes. In conclusion, the authors demonstrated the feasibility of their technique (prevascularized polymer device with hepatotrophic stimulation), using human hepatocytes. Further studies are underway, before implementation of human clinical trials.

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.

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 x 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 x 3 x 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.

Joint resurfacing with cartilage grown in situ from cell-polymer structures.

We tested the potential of a new technology developed in our laboratory to create new hyaline cartilage for resurfacing distal femoral joint surfaces of New Zealand White rabbits that had been surgically denuded of articular cartilage. We removed hyaline cartilage from the patellar groove of the distal femurs in 24 rabbits. Chondrocytes isolated from the excised cartilage of 12 of these rabbits (experimentals) were seeded onto synthetic biocompatible, biodegradable polymers composed of polyglycolic acid. The cells were labeled in vitro with a thymidine analog, BrdU (5-bromo-2'-deoxyuridine). After 1 week in vitro, the cell-polymer structures were implanted onto the denuded surfaces of 12 defects made in the hyaline cartilage of the contralateral knees of the experimental animals. Twelve control animals received either no implants or implants not containing cells on similar surgical defects. After 7 weeks, we found evidence of new cartilage growth in 11 of the 12 experimental animals and virtually no new cartilage formation in any of the animals in either control group. Immunohistochemical analysis demonstrated the presence of BrdU-labeled chondrocytes in representative specimens.

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.

Angiopolarity of cell carriers: directional angiogenesis in resorbable liver cell transplantation devices.

The purpose of this study was to obtain directional angiogenesis of small blood vessels and capillaries to an implant made from a resorbable polymer for hepatocyte transplantation. It was intended to mimic the native acinar structure of the liver in order to facilitate replication of the cells and organ growth. The implant device structure was designed for injection to minimize surgical trauma. Hollow microspheres with an open porous wall structure and one large central opening were made from poly(d,l-lactic-co-glycolic acid) (85:15 lactic:glycolic). This polymeric scaffold was seeded with hepatocytes and implanted into the abdominal wall muscle of syngeneic Fisher rats. Specimens explanted up to 56 days p.o. showed hepatocyte survival and the development of a directional blood supply. This phenomenon is coined "angiopolarity". The study should help in addressing the issue as to whether avascular cell implants with post-transplantation organ growth should be attempted. Processing options in applying heat to the polymer solution allow manufacturing of larger microspheres with different diameters of central openings. This would allow the use of the scaffold for other cell transplantations than hepatocytes.