Graft Interface Research Articles

Cages: designs and concepts

Many new interbody fusion cages have been recently developed, but clinical studies analyzing fusion outcome are still scarce. Radiological methods to assess fusion are not standardized and are often unreliable. Cages have been stated to provide good segmental distraction, provide axial load support and reduce segmental mobility, but there have been reports of failed fusions because of implant failure. This paper presents a critical opinion on current cage designs, stressing their clinical and biomechanical implications. Threaded cage designs compromise endplate integrity, and when placed in pairs have inherent limitations for distraction. Non-threaded cage designs usually preserve endplate integrity, but geometry may be inadequate to provide a good surface match to the endplate. The concept of an open frame type cage is believed to have biological advantages, because large graft volumes inside the cage can be in direct contact with host bone. Cadaveric tests suggest that open frame constructs have compressive strength similar to that of full surface contact cages. Restoration of segmental height, sagittal balance and increased neuroforaminal clearance are all functions of disc space distraction. The effect of cage instrumentation on axial load distribution, however, is not well understood. Biomechanical experiments strongly suggest supplementing cage instrumentation with posterior fixation, to achieve a marked increase in initial segmental stability. In the absence of gross segmental instability, micromotion at the host graft interface may still exist. As a result, fusion will never occur, instead a pseudoarthrosis will develop. For monitoring fusion, the use of non-metallic cages has distinct advantages, because no metal artifacts will disturb radiological assessment.

Insertion of autologous tendon grafts to the bone: a histological and immunohistochemical study of hamstring and patellar tendon grafts.

This study examined the structure of the insertion of autologous tendon grafts used for anterior cruciate ligament reconstruction. Biopsy specimens of the femoral paragraph signand tibial bone graft interface were obtained at revision surgery in 14 patients (6 with hamstring grafts, 8 with a patella tendon graft). The specimens were analyzed by light microscopy and immunohistochemistry (confirming type I, type II, and type III collagen). The insertions of hamstring autografts to the bone tunnel have three characteristic histological zones. Zone 1 is composed of the dense connective tissue of the graft. The collagen fibers of the graft enter the bone under oblique angles. Zone 2 is composed of woven bone with a sharp transition to the lamellar bone of the tibia (zone 3). Immunohistochemistry revealed the presence of type I and type III collagen within the dense connective tissue of the graft. The woven and lamellar bone showed positive immunostaining for antibodies against type I collagen only. This structure resembled a fibrous ligament or tendon insertion. In the majority of patients with a patella tendon graft the structure of the insertion resembled a chondral enthesis. The chondral insertion of the graft to the bone is composed of four characteristic zones. Between the dense connective tissue of the graft (zone 1) and bone (zone 4) there is a zone of fibrocartilage (zone 2). Close to the bone the fibrocartilage is mineralized (zone 3). Within the fibrocartilage the immunohistochemical analysis confirmed type II collagen. This structure resembled the chondral enthesis of the normal anterior cruciate ligament. However, in cases in which the distal bone bloc has been fixated outside the tibial tunnel, the tibial insertion of the patellar tendon graft resembled a fibrous insertion. While both types of tendon grafts heal to the bone of femur and tibia, the insertion of patella tendon grafts healing by bone plug incorporation resembles the chondral insertion of the normal anterior cruciate ligament and may have a more physiological connection to the bone than hamstring grafts.

Relation between phytohormone level and vascular bridge differentiation in graft union of explanted internode autografting

Development of the graft union of explanted internode autografting of cucumber (Cucumis sativus Linn.) culturedin vitro is regulated by plant hormones exogenously added to the media. The levels of auxin (indole-3-acetic acid, IAA) and zeatin plus zeatin riboside (Z + ZR) in graft unions and in 4 parts of the graft union have been analyzed by ELSA assay. Results show that vascular bundle regeneration in the graft union is co-regulated by IAA and Z + ZR. Differentiation of vascular bridge across the graft interface requires higher level of IAA and IAA/ (Z + ZR) ratio than that of anastomosing bundle, indicating that plant hormones are limiting and controlling factors for the development of graft union.

Callus formation in relation to changes of the membrane Ca2+ in the graft unions ofGinkgo biloba L.

The relationship between the callus formation and intracellular Ca2+ at the early stage of development of graft unions inGinkgo biloba L. was investigated. On the second day after grafting, part of the undamaged living cells at the graft interface of the stock and scion in the regions of the cortex and pith began to dedifferentiate or divide. 8 d after grafting, a great number of callus cells were produced from the cortex and phloem, while callus bridges linking the stock with scion formed between the callus cells of the graft partners from the cortex. A membrane Ca2+ probe, chlortetracycline (CTC), was used to locate the intracellular Ca2+ distribution and it was found that the cortical and pith parenchyma cells were the first to show remarkably increased CTC-Ca2+ fluorescence, suggesting that the cortical and other parenchyma cells play a leading role at the early stage of development of graft unions by earlier dedifferentiation and more rapid production of callus cells.

Early Scion/Stock Junction in Compatible and Incompatible Pear/Pear and Pear/Quince Grafts: a Histo-cytological Study

A comparative histo-cytological study of compatible and incompatible pear/pear and pear/quince grafts, grown in a glasshouse, was carried out on unions collected from day 1 to day 22 after budding (DAB). Successive steps leading to the graft junction were studied. Adhesion between the two partners was observed along the inner bark tissues (phloem and cambium) just 24 h after budding, while a necrotic layer appeared at the graft interface. The first cell divisions were observed 3 DAB. In the inner bark interface these led to the formation of a junction callus, inside which new cambial strands (neocambia) appeared between 10 and 15 DAB, starting from the cut edges of the scion and stock cambia, and joining later when they began to produce both phloem and xylem derivatives. Along the outer bark interface, where the cut surfaces of the bark parenchyma were exposed to the air, divisions of the parenchyma cells gave rise to a new phellogen layer (neophellogen) approx. 6 DAB, and suber production was observed 10 DAB. No obvious differences were noticed between compatible and incompatible grafts during the first stages of graft junction. No particular cell necrosis phenomenon was observed at the interface of incompatible grafts. Nevertheless, the neocambial junction appeared to be delayed in pear/quince heterografts, especially in incompatible ones. This could be the first structural event of the incompatibility response in pear/quince grafts.

Early Scion/Stock Junction in Compatible and Incompatible Pear/Pear and Pear/Quince Grafts: a Histo-cytological Study

A comparative histo-cytological study of compatible and incompatible pear/pear and pear/quince grafts, grown in a glasshouse, was carried out on unions collected from day 1 to day 22 after budding (DAB). Successive steps leading to the graft junction were studied. Adhesion between the two partners was observed along the inner bark tissues (phloem and cambium) just 24 h after budding, while a necrotic layer appeared at the graft interface. The first cell divisions were observed 3 DAB. In the inner bark interface these led to the formation of a junction callus, inside which new cambial strands (neocambia) appeared between 10 and 15 DAB, starting from the cut edges of the scion and stock cambia, and joining later when they began to produce both phloem and xylem derivatives. Along the outer bark interface, where the cut surfaces of the bark parenchyma were exposed to the air, divisions of the parenchyma cells gave rise to a new phellogen layer (neophellogen) approx. 6 DAB, and suber production was observed 10 DAB. No obvious differences were noticed between compatible and incompatible grafts during the first stages of graft junction. No particular cell necrosis phenomenon was observed at the interface of incompatible grafts. Nevertheless, the neocambial junction appeared to be delayed in pear/quince heterografts, especially in incompatible ones. This could be the first structural event of the incompatibility response in pear/quince grafts.

Phloem translocation in regeneratingin vitro- heterografts of different compatibility

Phloem translocation of [ 14 C]-sucrose and 5/6-carboxyfluorescein (CF) from scion into the stock was studied in in vitro-heterografts of Lycopersicon on Solanum (L/S) and Vicia on Helianthus (V/H) at various stages of regeneration. Autografts of all partners served as controls. Corresponding with the translocation experiments newly formed sieve-tube connections between the graft partners were counted. 14 C-translocation experiments with 14 C]-sucrose revealed an age-dependent increase of radioactivity in the stock of all combinations. In L/S and all autografts the major increase of 14 C-label in the stock occurred 5-10 d after grafting. In V/H, however, import of label into the stock remained low throughout the regeneration period. In L/S grafts, increases in the numbers of sieve-tube connections parallel the increasing rate of 14 C-transport, indicating functioning sieve-tube connections in the graft union. In contrast, V/H grafts did not show this strong correlation between structure and function of wound repair phloem. This suggested the existence of non-transporting sieve-tubes between the graft partners. Similar results were obtained with CF-transport, showing that effective phloem translocation across the graft interface occurred in L/S, but not in V/H grafts. The observed differences in phloem translocation are discussed with regard to compatibility/incompatibility phenomena in heterografts.

Insights into pigmentary phenomena provided by grafting and chimera formation in the axolotl.

The expression of pigmentation patterns in axolotl pigmentary mutants was observed following three types of experimental manipulations including chimera formation, reciprocal neural crest grafts, of gonadal primordia. Three pigmentary genes were utilized including the wild type (D), white (d), and albino (a). In chimeras between white and albino embryos, melanoblasts from the white half crossed the graft interface to differentiate in albino skin. Neural crest grafts from white embryos to albinos provided melanophores of white origin that were capable of differentiation in albino skin. Grafts of gonadal primordia from albino to white embryos provided albino germ cells that formed unpigmented ovocytes together with dark ovocytes: white ovocytes from the albino grafted ovary, and dark ovocytes from the host ovary. The donor albino white ectoderm included in the graft was able to support the differentiation of melanophores, iridophores, and xanthophores that invaded the graft ectoderm from the neural crest of the white host. It was concluded that manifestation of the white or wild phenotypes may be related to the possible presence or absence of inhibiting or stimulating pigmentary factors in the skin. This possibility was discussed in the light of recent discoveries of such factors as Agouti Signaling Protein (ASP) from mammalian skin.

Axons of Xenopus neural tube respond to reversals of neural tube orientation.

Axonal trajectories of the Kolmer-Agduhr (KA) neurons of Xenopus embryos, were observed after anterior-posterior (A-P) inversions of neural tube grafts to determine whether KA axons follow cell-inherent directional cues, cues from their immediate environment, or rostrocaudal signals from the embryo. KA axons form one of the earliest ascending spinal pathways in Xenopus and are visible in the lateral marginal zone of whole mounts processed for GABA immunoreactivity. Grafts were made at trunk levels at stages 22-24, 3-5 h before the first KA neurons were detectable and prior to axonal out-growth. Embryos were fixed and immunostained 6-36 h later. KA trajectories within and adjacent to reversed grafts were compared to those of nonrotated control grafts and to neural tube lengths comparable in position and in length in unoperated embryos. Most KA axons within rotated grafts followed the graft's orientation. However, others changed direction, taking novel routes, including turning to conform to the orientation of the host embryo. Reorientations were most common near the posterior host/graft interface. Some host KA cells also reoriented, always within a few hundred microns of the graft interface. Taken together, these growth patterns show that most KA axons within the grafts grow normally with respect to the original polarity of the graft neural tube and maintain that direction even into tissue of opposite polarity, suggesting that their routes are mainly determined by cell-intrinsic and/or local tissue factors. However, the reorientation of many other axons, particularly near graft seams, implies that KA axons can respond to local fluctuations in directional or segment identity signals generated in both host and graft after this perturbation.

Vascular Differentiation in the Graft Union of in-vitro Grafts with Different Compatibility. — Structural and Functional Aspects

Summary Various in-vitro grafts of explanted internodes - the heterografts Nicandra physaloides on Lycopersicon esculentum and Vicia faba on Helianthus tuberosus as well as autografts of Nicandra physaloides and Vicia faba - were established in sterile culture. The histology of the graft union was studied by light and electron microscopy at different stages of development following the grafting procedure. Callus proliferation at the graft union started mainly in close association with the cut vascular bundles, while in the pith region an isolation layer was formed. Wound vascular elements differentiated either directly from parenchyma or, as a rule, from callus cells 3–5 days after grafting. Continuous wound xylem and wound phloem reconnecting the cut vascular bundles of both partners across the graft interface were formed in Nicandra as well as in Vicia autografts after 5 days. In the interfamiliar graft Nicandra/Lycopersicon continuous phloem connections were found 7 days after grafting. Wound cambium giving rise to secondary phloem and xylem elements was established in Nicandra autografts 7–10 days following grafting. Functioning of the wound phloem across the graft union has been examined at different developmental stages of in-vitro grafts using 14C-sucrose. One to 3 days after grafting there was only a small import of labelled sucrose into the stock, likely due to symplasmic or apoplasmic diffusion across the callus of the graft union. The translocation rate increased with the onset of phloem regeneration about 5-7 days after grafting. In the less compatible Nicandra/Lycopersicon graft about 4 weeks after grafting the import into the stock decreased again and abnormal vascular complexes were found in the scion at the same time; finally the whole graft withered away. Incompatibility of the interfamiliar graft Vicia faba on Helianthus tuberoses was characterised by a complete loss of vascular regeneration between the scion and stock and - consequently - by an extremely low rate of 14C-translocation into the stock.

Preliminary experience with a novel model assessing In vivo mechanical strength of bone grafts and substitute materials

A novel canine tibia model was used to evaluate four bone graft materials: autologous cortical bone, allograft cortical bone, hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic granules, and a HA/TCP and collagen composite. Mechanical material properties were assessed using custom-designed stainless steel plugs for control of graft volume and interface surface area. These plugs held the bone graft materials in the cortex of the tibia shaft and allowed in vivo mechanical testing. After 6 months of ad lib weight bearing, the grafts were harvested and tested in torsion. The samples in each animal were compared with the test plugs into which new bone had grown without the addition of graft. Control bone peak shear strength averaged 47 (+/- 8.3) MPa (6.78 +/- 1.2 kpsi). Compared on the basis of peak torque, stiffness, and energy to peak torque, no significant differences were found among any of the graft materials or control bone. Histologic examination revealed the materials to be osteoconductive with the extensive formation of dense, compact cancellous bone. The new bone in the autograft and allograft samples completely filled the available space, whereas gaps persisted in the synthetic ceramics.

The Function of Phloem Connections in Regenerating In Vitro‐Grafts*

AbstractIn order to answer the question whether functioning phloem connections exist between graft partners, phloem transport has been studied in cultured explant‐grafts after application of 14C‐sucrose and carboxyfluorescein (CF) to the scion. Autografts of Lycopersicon esculentum and Helianthus annuus were investigated at various regeneration periods. Ungrafted internodes served as controls.A segmental analysis was used to determine the tissue distribution of 14C‐sucrose in a graft. The 14C‐profiles obtained show that sucrose translocation across the graft interface started 4 days after grafting and increased later. The observed translocation appears to occur via wound phloem, since at this time the first complete wound‐phloem bridges (shown as files of aniline‐blue‐positive sieve plates) traverse the graft interface. In 7‐d‐old autografts, sucrose transport across the graft interface returned to normal again, as indicated by the distribution of the label. In addition, 14C‐profiles reveal accumulation of label in sink tissues. Here the basal callus of the stock, and temporarily the graft union itself, represent the main sinks for labelled sucrose.Translocation of CF was analyzed in hand sections of the grafts. The beginning of translocation into the stock was confirmed with the dye. Moreover, effective phloem translocation across the graft interface, visualized with CF, could undoubtedly be assigned to wound‐phloem bridges reconnecting the cut vascular bundles of scion and stock. Thus, the function of phloem connections in regenerated in vitro‐grafts is directly shown.

The Structure and Composition of Bead-like Projections on Sitka Spruce Callus Cells Formed during Grafting and in Culture

Projections at the cell wall surface in callus formed at the graft interface and in vitro in Picea sitchensis (Bong.) Carr have been examined using light and electron microscopy. These structures range in size from 1 to 5 μm in diameter, and consist of a homogeneous matrix enclosing one or more fibrillar/vesicular cores. Histochemical and cytochemical studies have shown that the homogeneous matrix is made up of a mixture of pectins, carbohydrate, protein and fatty acids, while the fibrillar/vesicular component is mainly carbohydrate and pectins. The possible functions of these structures are discussed on the bases of their composition.

Electrical resistance as a measure of graft union

Measurement of electrical resistance across the graft interface of the autograft ( Lycopersicon esculentum ) is employed as a simple device for detecting the succes of graft unions, the accompanied structural changes at the graft junction being checked by light and electron microscopy for reference. Electrical resistance for the first 2 – 3 days increases rapidly in step with the formation and thickening of the isolation layer. In the next 3 – 8 days electrical resistance decreases steadily, as the isolation layer ruptures and disappears during callus proliferation and interdigitation. Secondary formation of plasmodesmata takes place between contacting cells of stock and scion, and differentiation of vascular elements appears in callus. Henceforth, resistance begins to drop to the level of the intact stem, which seems to indicate that symplastic connection and vascular unification have been completed. In contrast to tomato autografts, resistance measurements made across an incompatible heterograft ( Amaranthus tricolor/Lycopersicon esculentum ) exhibit a steady increase with the establishment of the isolation layer, which remains unruptured. The results are discussed in relation to graft compatibility/incompatibility and the possibility of application in commercial practice.

Studies on Graft Unions IV. Assimilate Transport and Sieve Element Restitution in Homo- and Heterografts

Summary Using 14 C-labelling techniques the rates of assimilate transport between scion and stock of the compatible intrafamiliar graft Lycopersicon esculentum on Solanum tuberosum and the less compatible interfamiliar graft Vicia faba on Helianthus annuus were compared. The homografts Lycopersicon/Lycopersicon, Helianthus/Helianthus and Vicia/Vicia served as controls. Corresponding with the transport experiments the phloem contacts in graft unions at different stages of development were examined. Assimilate transport across the graft interface started in the compatible heteroplastic and homoplastic combinations 5–7 days after grafting. In the less compatible Vicia/Helianthus grafts label from the scion could not be detected in the stock until 10 days after grafting. The maximum rate of assimilate transport from one partner to the other was significantly lower in Vicia/Helianthus grafts than in the other systems. The first, sieve tubes traversing the graft interface could be found in Lycopersicon/Solanum simultaneously with the appearance of assimilates in the stock. With further differentiation the increasing numbers of sieve tubes in the graft unions correlated with an increasing rate of transported assimilates, indicating that the route of translocation was via the phloem.

Application of a fluorescent dye to study connectivity between third ventricular preoptic area grafts and host hypothalamus.

The mutant hypogonadal (hpg) mouse lacks a functioning gene for the neurohormone gonadotropin releasing hormone (GnRH). Previous studies from our laboratory had indicated that the initiation and maintenance of reproductive function in these mice could be brought about by the implantation of normal fetal grafts into adult hosts. Testicular or ovarian growth and other indicators of normal neurosecretory output were always accompanied by survival of GnRH neurons and growth of GnRH axons into the host median eminence where such axons terminate on the hypophysial portal capillaries. To determine if other connections exist between graft and the host hypothalamus, small crystals of the carbocyanine dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) were applied to either graft or host after fixation of the brain. Tissue sections were analyzed for retrograde and and anterograde movement of the dye. When crystals were placed on the graft, labeled axons were found in the host median eminence or in the host hypothalamus taking an arching trajectory toward the median eminence. Retrogradely labeled neurons in the host were few in number and largely confined to the host arcuate nucleus. With DiI crystals applied to the basal hypothalamus, labeled axons were distributed widely in the host but much sparser in the graft. Axons appeared to enter primarily at sites where the graft and host interface lacked an ependymal lining. Small numbers of retrogradely labeled neurons were also seen in the graft. Most were cells of very simple morphology and were distributed randomly in the graft. When double label experiments were carried out most DiI positive cells in the graft contained GnRH. These results indicate the connectivity between host hypothalamus and the third ventricular preoptic area grafts exists but is limited in nature.

Studies on graft unions

De novo formation of cytoplasmic cell connections are studied at the graft interface of 5 day old in vitro heterografts ofVicia faba onHelianthus annuus. Continuous and half plasmodesmata, both branched and unbranched, are described at various stages of development in non-division walls between unlike and like dedifferentiated callus cells. In apical portions of protruding callus cells and in the contact zone between opposing cells extremely thin wall parts with a striking ER/plasmalemma contact are observed. During subsequent thickening of the modified wall parts cytoplasmic strands enclosing constricted ER cisternae are entrapped within the newly deposited wall material. These cytoplasmic strands represent half plasmodesmata which—in case of fusion with corresponding structures of adjoining cells across the loosened wall matrix — form continuous cell connections. Golgi vesicles secreting wall material are involved in the process of forming half and continuous plasmodesmata, thus following the same mechanism of plasmodesmata development as described for isolated protoplasts in cell cultures. The findings suggest the existence of a unifying mechanism of secondary formation of plasmodesmata showing far-reaching similarities with the establishment of primary cell connections.

Graft Union Development and Symplastic Phloem Contact in the Heterograft Cucumis sativus on Cucurbita ficifolia

Summary The compatible heterograft, Cucumis sativus on Cucurbita ficifolia , has been chosen to study the development of the graft union from the formation of an isolation layer and a parenchymatous callus at the graft interface up to the occurrence of symplastic contact between the cells, especially the sieve elements of stock and scion. Lacking species-specific ultrastructural markers, symplastic phloem contact between Cucumis and Cucurbita is demonstrated indirectly by serial sectioning. Phloem development in the graft unions results in different numbers of connecting sieve tubes in each individual graft, but the average number of sieve tube connections in Cucumis/Cucurbita is strikingly lower than in the respective homograft, Cucumis/Cucumis . The discussion focusses on the question, whether the observations suggest the existence of species-specific cell recognition and inherent compatibility/incompatibility, or whether non-species-specific substances, like phytohormones released by damaged vascular tissue, provide sufficient interspecific coordination to make the graft union develop and work in harmony.

Effects of tissue adhesive (Tisseel) on corneal wound healing in lamellar keratoplasty in rabbits.

The purpose of this study was to compare corneal wound healing between suture and sutureless lamellar keratoplasty in the rabbit eye. The tissue adhesive (Tisseel, Vienna) a commercially available two component adhesive system based on human fibrinogen, which is activated by thrombin, was used. 8.0mm half-thickness lamellar keratoplasties (autotransplants) were performed on the twenty-four rabbit eyes with tissue adhesive and the other eyes were operated with 10-0 nylon continuous suture as a control, respectively. At varying periods postoperatively, they were killed and the eyes were immediately enucleated and examined by transmission electron microscopy and light microscopy. The procedure was completed by the application of a bandage soft contact lens. Seventy-one percent of the glued lamellar keratoplasty grafts were retained and in six eyes, focal white opacities at the bed and graft interface were noted. There was no change in corneal contour, with the only irregularity being at the graft/host junction in the tissue adhesive group. Histopathologic examination of the glued eyes demonstrated the presence of a layer of eosinophilic material between the corneal bed and the lamellar graft but a minimal inflammatory response was observed in the early postoperative days. At fourteen days the adhesive was no longer visible and at three weeks, the graft was well healed. We believe that this study indicates a potential adjunctive role for tissue adhesive in corneal wound healing.

Development and Structure of Unusual Xylem Elements During Graft Union Formation in Picea sitchensis L.

Electron microscope studies of Sitka spruce have been undertaken to observe the sequence of cellular development following grafting. Observations at the graft interface reveal a recovery and regeneration sequence associated with union formation. Xylem elements differentiated directly from the vascular cambia of the rootstock and scion are different from elements arising from parenchymatous callus derived from ray parenchyma, which may be produced in an attempt to establish a connecting water conducting system as rapidly as possible. Failure to form such a system may be a primary cause of graft failure.