Ординатура / Офтальмология / Английские материалы / Corneal Endothelial Transplant (DSAEK, DMEK & DLEK)_John_2010

Abstract

Cellular organization of foreign grafts constructed from cultivated cells is critical to successful graft-host integration and tissue repair. This chapter described a novel human corneal endothelial cell (HCEC) therapeutic method, where cultivated adult HCEC sheet with uniform orientation was prepared and transplanted to a rabbit cornea. Having a correct morphology and intact barriers, the HCEC sheet was made by the temperature-modulated detachment of monolayered HCECs from thermo-responsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted surfaces and was delivered with proper polarity to the corneal posterior surface by a bioadhesive gelatin disk. Results of the in vivo studies, including the follow-up clinical observations and histological examinations showed the laminated HCEC sheet was successfully integrated into rabbit cornea denuded with endothelial layer after the biodegradation of gelatin carrier. These data indicate the feasibility of the proposed procedure in cell therapy for corneal endothelial cell loss.

Introduction

Human corneal endothelial cells (HCECs) maintain corneal clarity by means of a barrier function and pump-leak mechanism.1 Although the number of HCECs decreases with aging, ocular trauma/surgeries, contact lens wearing or inflammations, HCECs do not proliferate in vivo to compensate for the cell loss.2 In more than half of all global cases involving penetrating keratoplasty (PK, a full-

thickness corneal transplantation), the only corneal component that requires replacement is the endothelial cell layer. Given insufficient supplies of donor corneas and complications of PK, there would be a substantial advantage in being able to replace the endothelium alone by delivering cultured HCECs to the recipient.

Corneal endothelial cell transplantation was attempted to repopulate rabbit cornea with unhealthy endothelium by directly injecting a cell suspension into the anterior chamber.3 However, that trial was limited because of only scattered clumps of endothelial cells randomly attached to the targeted cornea and to other normal ocular tissues such as the iris and lens. In recent years, numerous investigators have reported a method to transplant corneal endothelial cells by seeding and cultivating them on different carriers made of either natural tissue materials4,5 or artificial polymeric materials.6-9 Although a monolayered architecture of cultured cells was maintained, the intraocular grafting of these engineered tissue replacements may possibly cause problems such as unstable attachment of the cell carrier membrane to the host corneal stroma and fibroblastic overgrowth between the membrane and stroma.3 By avoiding the permanent residence of foreign carrier materials in the host, our group has recently presented a novel cell sheet-based therapy for corneal endothelial reconstruction (Figure 38-1). Bioengineered

HCEC sheets were fabricated from thermoresponsive culture supports and were delivered by using multifunctional gelatin hydrogel disks.10-12 The advantage of this strategy is that it allows the HCEC sheet grafts with their deposited extracellular matrix (ECM) can be directly transplanted onto the damaged corneas without biomaterial barriers.

Cell sheet engineering is a novel technology for harvesting cultivated cell sheets via temperature modulation of thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)-grafted culture surfaces.13 By using this method, it is feasible to create transplantable tissue/organ sources without using a three-dimensional biomaterial scaffold, which may elicit host inflammatory responses after in vivo implantation of tissue-engineered replacements into damaged sites.14 Recently, this powerful technology has been proven to be effective for cardiac tissue repair15,16 and corneal epithelial reconstruction.17-19

It has been reported that cells could adhere and proliferate on the hydrophobic PNIPAAm-grafted surfaces at 37°C, and spontaneously detached from the switched hydrophilic surfaces when the culture temperature was reduced to a level below the lower critical solution temperature of PNIPAAm (i.e. 32°C in water).20 To harvest the cell cultures as whole sheets instead of as isolated suspensions, we have fabricated the PNIPAAm-grafted

culture supports by means of plasma chemistry, a unique technique that previously allowed us to develop artificial corneas.21-23 In this study, untransformed adult HCECs were cultivated on the nanostructured PNIPAAm-grafted surfaces for 3 weeks at 37°C, and confluent monolayers were obtained at 20°C (Figure 38-2). The characteristics of bioengineered HCEC sheets were determined in vitro by evaluating their viability and by scanning electron microscopy, immunohistochemistry, and histological studies. Evaluations of native corneal endothelium from human eye bank donors were conducted simultaneously for comparison.

After removal of the PNIPAAm-grafted surfaces at a low culture temperature, the HCEC sheets are usually soft and fragile due to the loss of anchorage dependence. It may be necessary to apply supporting materials to strengthen these cell monolayers for transportation and surgical handling. Gelatin is obtained by thermal denaturation or physical and chemical degradation of collagen. It has been reported that a gelatin membrane substrate cross-linked with glutaraldehyde could be used to support the growth of cultivated rabbit corneal endothelial cells for transplantation.6 Our previous study has also demonstrated the feasibility of fabricating native gelatins into sandwich-like encapsulating membranes for retinal sheet transplantation.24 In this investigation, given the bioadhesive and

prompt degradable properties, gelatin disks were used for the delivery of HCEC sheet to the corneal posterior surface, the denuded Descemet’s membrane (i.e. the basement membrane of corneal endothelium). We performed an in vivo study in a rabbit model to examine the efficacy of the use of bioengineered HCEC sheets for improving corneal endothelial reconstruction.

Experimental

Materials

N-isopropylacrylamide (NIPAAm) was purchased from Acros (Fairlawn, NJ, USA). OPTI-modified Eagle’s medium (OPTI-MEM), Hank’s balanced salt solution (HBSS, pH 7.4), gentamicin, and trypsin-EDTA were obtained from Gibco BRL (Grand Island, NY, USA). Dispase II was purchased from Roche Diagnostics (Indianapolis, IN, USA). Fetal bovine serum (FBS) and antibiotic/antimycotic (A/A) solution were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). Human recombinant epidermal growth factor (EGF) was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Nerve growth factor (NGF) was obtained from Biomedical Technologies (Stoughton, MA, USA). Pituitary fibroblast growth factor (FGF), ascorbic acid, chondroitin sulfate, calcium chloride, human lipid fraction, RPMI 1640 vitamin solution, and PKH26 red fluorescent dye were purchased from SigmaAldrich (St. Louis, MO, USA). All the other chemicals were of reagent grade and used as received without further purification.

Preparation of Thermoresponsive Culture Surfaces

Polyethylene (PE) dish substrates (35 mm in diameter) were ultrasonically cleaned in ethqanol for 1 hour before use. The detailed procedure for the plasma modification process has been described in a previous work.25 Briefly, a model PD-2 plasma deposition reactor with a bell jar-type glow discharge cell (Samco, Kyoto, Japan) was used. Under argon atmosphere, plasma was generated to activate the surfaces of PE substrates. The samples were immersed in a 10 wt% NIPAAm monomer aqueous solution. Photografting polymerization of NIPAAm onto the peroxidized sample surfaces was performed by ultraviolet light irradiation. The modified surfaces were washed for 3 days with cold deionized water to remove the NIPAAm homopolymers and dried under nitrogen atmosphere.

An atomic force microscopy (AFM) (Veeco Digital, Santa Barbara, CA, USA) was also utilized to scan surface

topography. All measurements were made in tapping mode with a silicon cantilever and a scan rate of 0.6 Hz. AFM images were recorded with a scan size of 1 µm and a data scale of 30 nm. Three measurements were done on different surface sites to calculate the mean surface roughness for each sample.

Preparation of Gelatin Hydrogel Disks

Gelatin (Nitta Gelatin, Osaka, Japan), manufactured through an alkaline process of bovine bone collagen, was used to prepare the multi-functional hydrogel carriers for HCEC sheet transfer. The isoelectric point (IEP), weightaverage molecular weight (MW), and polydispersity index of the gelatin sample, reported by the manufacturer are respectively 5.0, 100 kDa, and 2.3. Gelatin hydrogel disks (7 mm in diameter, 700-800 µm thick) were prepared by solution casting methods as described elsewhere.11 Briefly, an aqueous solution of 10 wt% gelatin was cast into a polystyrene planar mold, and air-dried for 3 days at 25°C to obtain hydrogel sheets. Using a corneal trephine device, the hydrogel sheets were cut out to create small gelatin disks (0.4 cm2, 700-800 µm thick). Afterwards, the gelatin disks were sterilized by gamma irradiation using a cobalt60 source located at the National Tsing Hua University (Hsinchu, Taiwan, ROC). According to our earlier report,24 irradiation was performed in the presence of air at a dose of 16.6 kGy, applied at a dose rate of 0.692 kGy/h; irradiation temperature, 25 ± 1°C.

Cell Preparation

This study adhered to the tenets of the Declaration of Helsinki involving human subjects and was approved by Institutional Review Board. Twenty-five corneas from human donors (age, 55-80 years) stored in Optisol-GS at 4°C were obtained from National Disease Research Interchange (Philadelphia, PA, USA). Primary culture of adult HCECs was performed in our laboratory as described elsewhere.12 Briefly, the corneal endothelium-Descemet’s membrane complex was digested using a 1.2 U/mL of dispase II in HBSS for 1 hour at 37°C. Afterwards, the solution was collected and centrifuged, and the resulting HCEC pellet was resuspended and cultured in growth medium containing OPTI-MEM, 15% FBS, 40 ng/mL of FGF, 5 ng/mL of EGF, 20 ng/mL of NGF, 20 µg/mL ascorbic acid, 0.005% human lipids, 0.2 mg/mL of calcium chloride, 0.08% chondroitin sulfate, 1% RPMI 1640 vitamin solution, 50 µg/mL of gentamicin, and 1% A/A solution. By this method, around 3 × 103 to 105 cells could be acquired from one donor cornea.

Cultures were then incubated in a humidified atmosphere of 5% CO2 at 37°C. Medium was changed every other day. After 1 week in culture, confluent cell monolayers were subcultured by treating with trypsin-EDTA for 2 minutes, and seeded at a 1:3 split ratio. Only secondpassage HCECs were used during all experiments.

Harvest of HCEC Sheets from Thermoresponsive Culture Supports

For the purpose of in vivo tracking, HCECs were labeled with PKH26 red fluorescent dye following manufacturer’s instructions.26 Thermoresponsive supports grafted with PNIPAAm at an optimal density of 1.6 µg/cm2 were used in this study. After surface sterilization with ultraviolet light for 2 hours in the laminar flowhood,27 HCECs were plated on PNIPAAm-grafted culture dishes at a density of 4 × 104 cells/cm2 and cultivated under the same conditions as mentioned for cell preparation. Cell morphology was observed by inverted phase-contrast microscopy (Nikon, Melville, NY, USA). To estimate the cell density, a micrometer scale was used to determine area for calculation of endothelial cell numbers in confluent cultures after 3 weeks of incubation. Six regions on each culture surface were randomly selected and cell nuclei within each area were counted manually at 40× magnification. For the harvest of cell sheets, the thermoresponsive supports containing confluent cultures were rinsed twice with warmed phosphate-buffered saline (PBS) and replenished with serum-free OPTI-MEM. The HCEC monolayers were detached from PNIPAAm-grafted surfaces by changing the culture temperature from 37°C to 20°C.

Viability Bioassay

Cell viability of harvested HCEC monolayers was determined by a membrane integrity assay, using the LIVE/ DEAD Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR, USA), which contains calcein acetoxymethyl and ethidium homodimer-1. Briefly, after washing three times with PBS, the HCEC sheets were stained with a working reagent, which is composed of 4 µL of ethidium homodimer-1, 2 mL of PBS, and 1 µL of calcein acetoxymethyl. The samples were incubated for 30 minutes at 37°C and viewed under an inverted fluorescence microscope (Eclipse TS100 equipped with an epifluorescence attachment; Nikon).

glutaraldehyde in 0.1 M cacodylic acid buffer (pH 7.4) overnight at 4°C. After rinsing with 0.1 M cacodylic acid buffer three times for 10 minutes each time, the specimens were post-fixed in 1% osmium tetroxide for 30 minutes and dehydrated in ethanol solutions 50%, 70%, 90%, and 100%, twice and during 10 minutes for each concentration. The samples were further dried with CO2 in a critical point dryer (Hitachi, Tokyo, Japan), and gold coated by ion sputtering (Structure Probe, PA, USA) before examination under SEM (Jeol, Tokyo, Japan) at an accelerating voltage of 10 kV.

Immunohistochemistry

Control samples and HCEC sheets were fixed with 4% paraformaldehyde for 10 minutes at 4°C. After washing with PBS, the fixed specimens were then permeabilized in 0.3% Triton X-100 for 15 minutes, and blocked with 4% bovine serum albumin in PBS for 30 minutes. The samples were incubated with primary antibodies overnight at 4°C in a moist chamber. The antibodies, diluted in PBS containing 4% bovine serum albumin, were directed against zonula occludens-1 (ZO-1) (1:100; Zymed Laboratories, South San Francisco, CA, USA) or Na+,K+-adenosine triphosphatase (ATPase) (1:150; Upstate Biotechnology). The negative controls were incubated without a primary antibody. The specimens were washed in PBS, and incubated with fluorescein (FITC)-conjugated or rhodamine (TRITC)-conjugated donkey anti-mouse IgG secondary antibodies (1:200; Chemicon International, Temecula, CA, USA) for 2 hours at room temperature in the dark. Unbound excess labels were removed by rinsing in PBS. The samples were viewed under fluorescence microscopy (Axioplan 2; Carl Zeiss, Oberkochen, Germany or BX51; Olympus, Tokyo, Japan).

Histology

Control samples and HCEC sheets were mounted onto precooled chucks in OCT embedding medium (Tissue-Tek, Sakura Finetek, Torrance, CA, USA) and frozen at -70°C. Frozen specimens were cut into 5-µm sections at -20°C using a cryostat. After the fixation with 4% paraformaldehyde for 1 minute, the sections were stained with 4’,6-diamidino- 2-phenylindole (DAPI; Vector, Peterborough, United Kingdom) for visualization of cell nuclei and examined using a fluorescence microscope (Axioplan 2; Carl Zeiss).

Whole corneas from human eye bank donors (control groups) and detached HCEC sheets were fixed with 2%

Animals were treated according to the ARVO (Association for Research in Vision and Ophthalmology) Statement for

the Use of Animals in Ophthalmic and Vision Research. Eighteen New Zealand white rabbits (National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, ROC) weighing 3.0 to 3.5 kg were used in this study. Surgery was performed in the single eye of animals, with the normal fellow eye.

As previously described, rabbit corneal endothelium was treated with 0.1 mg/mL of mitomycin-C (Sigma-Aldrich, St. Louis, MO) for 2 weeks to establish an animal model mimicking human corneas.10 For HCEC sheet transplantation, the rabbits were anesthetized intramuscularly with 10 mg/kg body weight of xylazine hydrochloride (Chanelle, Loughrea, Co. Galway, Ireland) and 60 mg/kg body weight of ketamine hydrochloride (Merial, Lyon, France), and topically with two drops of 0.5% proparacaine hydrochloride (Alcon-Couvreur, Puurs, Belgium). After disinfection and sterile draping of the operation site, the pupil was dilated with one drop of 1% atropine sulfate (Oasis, Taipei, Taiwan, ROC), and a lid speculum was placed. In the right eye of each rabbit, the cornea was penetrated near the limbus with a slit knife under the surgical microscope (Carl Zeiss, Oberkochen, Germany). The central 7 mm of corneal endothelium was removed using a silicone-tipped cannula. Once detached and floating in the medium, the HCEC sheets were immediately attached with the gelatin disks (cell apical side up). The gelatin-HCEC sheet constructs were subsequently implanted in the anterior chamber of the eye (cell apical side down) through a 7.5 mm sclerocorneal incision (HCEC sheet group, n = 6). The incision site was closed with 10-0 nylon sutures. Traumatized rabbit corneas received gelatin disk transplantation only (gelatin group, n = 6) or no transplantation (wound group, n = 6) were the controls.

After surgery, 1% chlortetracycline hydrochloride ophthalmic ointment (Union Chemical & Pharmaceutical, Taipei, Taiwan, ROC) was immediately applied to the ocular surface in all three groups. For topical administration of corticosteroids, each surgical eye received two drops of 0.3% gentamicin sulfate ophthalmic antibiotic solution (Oasis, Taipei, Taiwan, ROC) and one drop of 1% prednisolone acetate ophthalmic steroid suspension (Allergan, Westport, Co. Mayo, Ireland) four times a day during the follow-up.

Postoperative Evaluations

The corneal conditions were examined daily for 1 month postoperatively. Corneal clarity was assessed using slitlamp biomicroscopy (Topcon Optical, Tokyo, Japan). Central corneal thickness (CCT) was determined using an ultrasonic pachymeter (DGH Technology, Exton, PA, USA).

An average of ten readings was taken. The results of CCT were expressed as mean ± standard deviation (SD). Comparative studies of means were analyzed using Student’s t-test (two-tailed) with a statistical significance at p < 0.05.

At 2 weeks postoperatively, the surgical corneas of the three groups were excised. The integrity of tight junctions of grafted HCEC monolayers were studied by flat-mount preparations. Immunostaining of ZO-1 was performed as mentioned above. For histological examinations, the corneal samples were mounted onto pre-cooled chucks in OCT embedding medium and frozen at –70°C. Frozen specimens were cut into 5 µm sections at –20°C. After the fixation with 4% paraformaldehyde, the sections were stained with hematoxylin and eosin (H&E) and examined using a microscope equipped with fluorescence (Carl Zeiss).

Results

Harvest of HCEC Sheets from Thermoresponsive Culture Supports

Representative AFM images of the PNIPAAm-grafted PE surfaces are shown in Figure 38-3. The surfaces of PNIPAAm-grafted substrates became rougher after covering with grafted PNIPAAm homogeneously. The mean surface roughness of PNIPAAm-grafted samples was determined to be 14.6 ± 3.2 nm, indicating the PNIPAAm coating on the culture surface is nanostructured.

Figure 38-3: Representative topographic image of PNIPAAm-grafted surface was observed by tapping-mode atomic force microscopy (scan size = 1 µm, data scale = 30 nm). The mean surface roughness of nanostructured supports (n = 3) with a homogeneous covering of PNIPAAm was determined to be 14.6 ± 3.2 nm.

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Figures 36-4A to C: Phase-contrast micrographs of HCEC cultures on the thermoresponsive supports. (A) At 4 hours after seeding, the HCECs were attached on the PNIPAAm-grafted surfaces. (B) After incubation at 37°C for 3 weeks, a fully confluent endothelial monolayer was consisted of small, polygon-shaped cells. (C) At 20°C, the detachment of monolayered HCECs exhibited a sheet-like movement. Scale bars, 100 µm.

After 4 hours of plating, isolated HCECs attached and spread well on the PNIPAAm-grafted surfaces (Figure 38-4A). Cells grew readily to reach confluence after 1-week cultivation at 37°C. By a further incubation for 2 weeks in medium, a thick layer of extracellular matrix (ECM) was deposited at the basal cell surface to allow the formation of a fully confluent monolayer. Under a phase-contrast microscope, confluent HCECs on the PNIPAAm-grafted surfaces showed a generally polygonal morphology and a high cell density, around 2500 cells/mm2 that was almost the same as found in vivo (Figure 38-4B). This indicated that the ex vivo proliferation rate of HCECs on the thermoresponsive supports was maintained. By lowering the culture temperature to 20°C, the detachment of monolayered HCECs from the switched hydrophilic PNIPAAm-grafted surfaces forms a sheet-like movement (Figure 38-4C). During the sheet-like movement, each endothelial cell at the leading edge assembles by contracting fan-shaped lamellipodia. In addition, cell release from the PNIPAAm-grafted surfaces was observed (Figure 38- 5). At the beginning of the low-temperature treatment, the HCEC monolayers were rolled up at the margin of the culture surfaces and centripetally detached owing to the gradual hydration of the PNIPAAm-grafted chains (Figure 38-5A). Such a process of cell separation from thermoresponsive supports is a mode of sheet-like movement (Figure 38-5B). After 45 min of incubation, a laminated HCEC sheet with a size of around 0.75 cm2 was harvested from completely hydrated PNIPAAm-grafted surfaces, and was wrinkled because of a contracting force of this cell lamella (Figure 38-5C). These results suggest that the cell sheet detachment from thermoresponsive supports correlates closely with temperature-modulated surface wettability.

Viability Bioassay

Cell viability of harvested HCEC monolayers from PNIPAAm-grafted surfaces was determined by the LIVE/ DEAD Viability/Cytotoxicity assay (Figure 38-6). It depends on the intracellular esterase activity to identify the living cells, which cleaves the calcein acetoxymethyl to produce a green fluorescence. In dead cells, ethidium homodimer-1 can easily pass through the damaged cell membranes to bind to the nucleic acids, yielding a red fluorescence. Nearly all of the cells were vital throughout the central region of detached HCEC sheets (Figure 38-6A). Only very few dead cells were interspersed between the live cells of endothelial monolayers. However, the results for the peripheral region of HCEC sheets showed a large number of green-stained cells and red-stained nuclei in the margin of cell monolayers (Figures 38-6B and C). This was probably due to the loss of

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Figures 38-5A to C: Gross observations of cultured HCEC monolayer detachment from thermoresponsive supports after incubation at 20°C for 10 minutes (A), 25 minutes (B), and 45 minutes (C). Scale bars, 5 mm.

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Figures 38-6A to C: Cell viability of harvested HCEC monolayers was determined by staining with a LIVE/DEAD Viability/Cytotoxicity Kit in which the live cells fluoresce green and the dead cells fluoresce red.

(A) Merged green and red fluorescence image (central region of cell sheets). (B and C) Color-separated images (peripheral region of cell sheets). Scale bars, 100 µm.

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Figures 38-7A to C: Scanning electron microscopy (SEM) micrographs of human eye bank corneas (control samples) and HCEC sheets.

(A) Native endothelium in whole corneas showed a normal hexagonal cell shape with minor irregularities. (B) Within HCEC sheets, cells exhibited a polygonal morphology with multiple cellular interconnections (arrow). (C) A layer of extracellular matrix (ECM) (arrow) was deposited at the basal cell surface of cell sheets. Scale bars, 50 µm.

anchorage-dependence for marginal cells caused by overgrowth of a fully confluent cell sheet during 3 weeks of culture. These data clearly demonstrated that the majority of monolayered HCECs remained viable after detachment at 20°C, suggesting the low-temperature incubation does not compromise cell viability.

Scanning Electron Microscopy

SEM studies examined the morphological characteristics of native endothelium in human eye bank corneas and of monolayered cells in cultured HCEC sheets (Figure 38-7). In control groups, HCECs on the Descemet’s membrane from the eye bank corneas (donors = 50 years of age) packed together and formed a single continuous monolayer (Figure 38-7A). The individual cells were intact, had distinct borders, and possessed a hexagonal morphology with minor irregularities. By contrast, after immediate separation from thermoresponsive supports, the HCEC monolayers remained well-organized. The monolayered cells within HCEC sheets exhibited a polygonal phenotype and had multiple cellular interconnections (Figure 38-7B). The absence of clear boundaries between these single cells was probably due to the cell contraction caused by detachment at a low culture temperature. In addition, a thick ECM layer was observed on the basal cell surface of HCEC sheets

(Figure 38-7C).

Immunohistochemistry

Immunohistochemical staining of ZO-1, a tight junctionassociated protein, was used to determine whether the cells within HCEC sheets formed tight junctions. In human donor corneas, the cells of native endothelium retained the tight junctions that are responsible for establishing the passive permeability properties of the endothelial barrier (Figure 38-8A). Similar to that of control samples, ZO-1 was located at the cell boundaries of HCEC sheets suggesting the formation of focal tight junctional complexes (Figure 38-8B).28 At higher magnification, the discontinuity of ZO-1 localization in HCEC sheets, which is a normal feature in corneal endothelium, was observed with gaps occurring at the Y-junctions between three adjacent cells (Figure 38-8C). In addition, there was no evidence of apoptotic cell death in the HCEC monolayers after detachment via thermal stimulus (i.e. the lowtemperature incubation), as reflected by the maintenance of the integrity of cell nuclei (Figure 38-8D). On the other hand, Na+,K+- ATPase, an integral membrane protein complex responsible for regulating ionic pump functions, was located at the basolateral membrane of the HCECs within both control samples and detached cell sheets (Figures 38-8E and F).29