Julie R. Fuchs

Fetal tracheal augmentation with cartilage engineered from bone marrow-derived mesenchymal progenitor cells

BACKGROUND/PURPOSE The authors have described previously the use of engineered fetal cartilage in a large animal model of fetal tracheal repair. This study was aimed at comparing cartilage engineered from bone marrow-derived stromal cells (BMSC) to native and engineered cartilage, in this model. METHODS Ovine BMSC were expanded in vitro, seeded onto biodegradable scaffolds, and maintained in transforming growth factor beta 1 (TGF-beta1)-supplemented medium for 3 months (group I). Identical scaffolds were seeded with fetal chondrocytes (group II). All constructs were analyzed in vitro, implanted into fetal tracheas, and harvested after birth for further analysis. RESULTS There were no differences in survival between the groups. All BMSC-based constructs exhibited chondrogenic differentiation. Matrix analyses in vitro showed that both groups had similar levels of glycosaminoglycans (GAG) and type II collagen (C-II), but lower levels of elastin when compared with native fetal cartilage. Yet, compared with group II, group I had higher levels of GAG, equal levels of C-II, and lower levels of elastin. However, remodeling resulted in no differences between the 2 groups in any of these variables in vivo. CONCLUSIONS The bone marrow may be a useful cell source for cartilage engineering aimed at the surgical repair of severe congenital tracheal anomalies, such as tracheal atresia and agenesis, in utero.

Cartilage Engineering from Ovine Umbilical Cord Blood Mesenchymal Progenitor Cells

We aimed to determine whether three‐dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n = 4) harvested at 80–120 days of gestation by low‐density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n = 28) were maintained in a rotating bioreactor with serum‐free medium supplemented with transforming growth factor‐β1 for 4–12 weeks. Similar constructs seeded with fetal chondrocytes (n = 13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n = 10). Statistical analysis was by analysis of variance and Students t‐test (p < .01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix‐specific staining. In the CB constructs, there was a significant time‐dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type‐II collagen (C‐II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C‐II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C‐II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.

Fetal tissue engineering: in vitro analysis of muscle constructs

BACKGROUND/PURPOSE This study was aimed at examining the impact of different tissue engineering techniques on fetal muscle construct architecture. METHODS Myoblasts from ovine specimens of fetal skeletal muscle were expanded in culture and their growth rates determined. Cells were seeded at different densities onto 3 scaffold types, namely polyglycolic acid (PGA) treated with poly-l-lactic acid (PLLA), a composite of PGA with poly-4-hydroxybutyrate (P4HB), and a collagen hydrogel. Constructs were maintained in a bioreactor and submitted to histologic, scanning electron microscopy, and DNA analyses at different time-points. Statistical analysis was by the likelihood ratio and paired Students t tests (P <.05). RESULTS Fetal myoblasts proliferated at faster rates than expected from neonatal cells. Cell attachment was enhanced in the PGA/PLLA matrix and collagen hydrogel when compared with the PGA/P4HB composite. Necrosis was observed at the center of all constructs, directly proportional to cell seeding density and time in the bioreactor. CONCLUSIONS Fetal myoblasts can be expanded rapidly in culture and attach well to PGA/PLLA, as well as collagen hydrogel but less optimally to PGA/P4HB. Excessive cell seeding density and bioreactor time may worsen final construct architecture. These findings should be considered during in vivo trials of muscle replacement by engineered fetal constructs.

Fetal tissue engineering: chest wall reconstruction.

BACKGROUND/PURPOSE This study was aimed at applying fetal tissue engineering to chest wall reconstruction. METHODS Fetal lambs underwent harvest of elastic and hyaline cartilage specimens. Once expanded in vitro, fetal chondrocytes were seeded onto synthetic scaffolds, which then were placed in a bioreactor. After birth, fetal cartilage constructs (n = 10) were implanted in autologous fashion into the ribs of all lambs (n = 6) along with identical, but acellular scaffolds, as controls (n = 6). Engineered and acellular specimens were harvested for analysis at 4 to 12 weeks postimplantation. Standard histology and matrix-specific staining were performed both before implantation and after harvest on all constructs. RESULTS Regardless of the source of chondrocytes, all fetal constructs resembled hyaline cartilage, both grossly and histologically, in vitro. In vivo, engineered implants retained hyaline characteristics for up to 10 weeks after implantation but remodeled into fibrocartilage by 12 weeks postoperatively. Mononuclear inflammatory infiltrates surrounding residual PGA/PLLA polymer fibers were noted in all specimens but most prominently in the acellular controls. CONCLUSIONS Engineered fetal cartilage can provide structural replacement for at least up to 10 weeks after autologous, postnatal implantation in the chest wall. Fetal tissue engineering may prove useful for the treatment of severe congenital chest wall defects at birth.

In vitro cartilage regeneration from proliferated adult elastic chondrocytes.

The purpose of this study was to investigate cellular feasibility in the proliferation and differentiation status of adult chondrocytes for cartilage regeneration in comparison to fetal chondrocytes. Primary cells were isolated from adult (n = 6) and fetal (n = 6) sheep ear cartilages and expanded in 10% fetal bovine serum (FBS) containing Hams F12 medium, in which adult and fetal cell proliferation rates were compared using a WST-1 assay kit. Approximately 4 million cells were seeded onto each 1×1×0.2-cm (200 μL) nonwoven fabric scaffold made from polyglycolic acid. Cell/polymer constructs were cultured in serum-free DMEM/F12 medium supplemented with 5 ng/mL TGF-β2 and 5 ng/mL des(1-3)IGF-I (adult chondrocytes, group A) or in 10% FBS containing Hams F12 medium (adult chondrocytes, group B, and fetal chondrocytes, group C) as controls in a rotating bioreactor for 6 weeks. The proliferation assay showed that fetal cells had a significantly better growth potential than did adult cells. Histology and extracellular matrix analyses revealed that groups A and C qualitatively displayed better matrix deposition than did group B. In conclusion, although adult sheep elastic chondrocytes had less growth potential than did fetal cells, the serum-free medium supplemented with growth factors significantly enhanced the production of cartilage matrix secreted from proliferated adult sheep elastic chondrocytes.

Fetal hepatic haematopoiesis is modulated by arterial blood flow to the liver.

We describe an as yet unrecognised relationship between fetal hepatic haematopoiesis and arterial blood flow to the liver. To increase hepatic arterial flow, the common bile duct (CBD) was ligated in fetal lambs. Reduction of hepatic arterial flow was accomplished in age‐matched animals by hepatic artery (HA) ligation. Multiple analyses performed before term showed a significant increase in haematopoietic cell density in CBD animals when compared with sham controls and HA animals. In contrast, HA animals demonstrated a decrease in liver haematopoietic activity. Fetal hepatic haematopoiesis is dependent upon arterial blood flow to the liver.

504 FETAL HEPATIC ARTERY LIGATION SUPPRESSES LIVER HEMATOPOIESIS IN UTERO.

Introduction Fetal hematopoiesis involves a successive interchange of progenitor cell activity among different anatomical sites, with the liver playing a pivotal role in the sequence. The mechanisms that regulate this unique process may hold the clues to the treatment and possibly cure of numerous hematological and non-hematological diseases, both before and after birth. We hypothesized that fetal hepatic hematopoiesis is controlled by arterial blood flow to the liver. This study was aimed at determining the effects of changes in hepatic artery flow upon liver hematopoiesis in utero. Methods The experimental design was validated by previous data demonstrating (1) the negligible role of arterial blood flow on fetal hepatic oxygenation and (2) an increase in hepatic arterial flow as a consequence of biliary obstruction. Fetal lambs (n = 15) were divided into 3 groups at 106-113 days gestation (term = 145 days). Group I (n = 5) underwent ligation and division of the right and left hepatic arteries; group II (n = 4) underwent ligation and division of the common bile duct; and group III (n = 6) were sham-operated controls. Euthanasia was performed at comparable gestational ages near term, 23.7 ± 6.4 days postoperatively. Animals were blindly assessed for hepatic hematopoietic cell/island density by both standard histology and transferrin receptor (CD71) immunohistochemistry. Additional analyses included hepatic angiography, peripheral blood hemoglobin (Hb) content, and colony-forming unit (CFU) assays of hematopoietic progenitors. Statistical comparisons were by non-parametric and mixed model regression analyses, as appropriate (p < .05). Results Overall fetal survival was 93%. In group I, post-mortem arteriograms confirmed complete occlusion of the hepatic arteries, albeit with variable local collateralization. There was a significant reduction in the density of hematopoietic progenitors in group I when compared to groups II and III. This was further confirmed by a significant reduction of CFU in group I. Group II had a significantly higher density of hematopoietic progenitors than the other two groups. Fetal Hb levels were lower in group I when compared to groups II and III, but this did not reach statistical significance in this series. Conclusions Fetal hepatic hematopoeisis is dependent on arterial blood flow to the liver. These data demonstrate a unique and previously unrecognized role of the hepatic artery in fetal development. Fetal hepatic artery ligation is a novel surgical model for the study of fetal hematopoiesis.