One-pot synthesis of chitosan-g-(PEO-PLLA-PEO) via "click" chemistry and "SET-NRC" reaction

For the development of biocompatible and degradable biomaterials, a kind of well-defined graft copolymer consisting of chitosan back-bone and amphiphilic PEO-PLLA-PEO branch chains was synthesized by Cu(0) catalyzed one-pot strategy combining "click" chemistry and single electron transfer-nitroxide radical coupling (SET-NRC) reaction. First, the precursors of 6-azide-N-phthaloyl-chitosan, TEMPO-PEO-alkyne and mPEO-PLLA-Br were designed and produced. Then, the one-pot coupling reactions between these precursors were performed in the presence of nanosized Cu and PMDETA. The efficiencies of the coupling reactions were greater than 90% determined by the FTIR and ESR spectra. The structure of graft copolymer with 43% of the grafting ratio was confirmed by the spectral analysis. This work provided a route to prepare chitosan graft copolymer.     

Synthesis of well-defined graft copolymers by combination of enzymatic polycondensation and "click" chemistry

Aliphatic polyesters having pendant azide groups were prepared by enzymatic polycondensation in the presence of lipase from Candida antarctica type B (CAL-B). The grafting reaction to the N(3)-functional polyester was carried out quantitatively at room temperature using copper-catalyzed azide-alkyne cycloaddition (CuAAC, "click" reaction) with monoalkyne-functional poly(ethylene oxide) (alkyne-PEO, M(n) = 750 g/mol). Furthermore, both enzymatic polycondensation and "click" reaction were carried out successfully in sequential one-pot reaction. The graft copolymer was surface-active and self-assembled in water. The graft copolymer had a critical aggregation concentration (cac) of 3 × 10(-2) μM in water determined by surface tension measurements. Above cac, the graft copolymer formed single chains and aggregates having a hydrodynamic radius of ～75 nm. Furthermore, the surface activity of the polymers at the air-water interface was studied by Langmuir trough measurements. The Langmuir isotherm of the graft polymer showed a pseudoplateau resulting from desorption of PEO chains into the subphase upon compression.     

The "double barrel" free vascularized fibular bone graft

A further modification of the free vascularized fibular bone graft is described in which a transverse osteotomy is made from the anterolateral aspect of the fibular shaft just distal to the entry of the nutrient artery. This produces two vascularized bone struts that may be folded parallel to each other but that remain connected by the periosteum and muscle cuff surrounding the peroneal artery and vein. The proximal strut is vascularized by both a periosteal and an endosteal blood supply, whereas the distal strut is vascularized by a periosteal blood supply alone. This so-called "double barrel" free vascularized fibular graft has been employed in three patients with segmental bone defects of the distal femur and in one patient with adjacent bony defects of the radius and ulna.     

Reconstruction of the medial half of the lower eyelid using a "switch" split-lid procedure

Reconstruction of the medial half of the lower eyelid has one major disadvantage: It produces a scar at right angles to the eyelid rim. In contrast, use of a "switch" split-lid procedure avoids this inconvenience. The lateral half of the lower eyelid is split in two lamellae. The inner layer is transferred medially, and the resulting defect is closed with a buccal graft. The outer layer is drawn laterally to cover the raw surface of the mucosal pedicle and graft. The surplus of skin over the lateral canthal area is removed. This procedure, which so far has been used in three patients, promises to be a useful alternative for reconstruction of the medial half, but not more, of the lower eyelid.     

Microsurgical forearm "cricket bat-transformer" phalloplasty.

Presently, the donor flap of choice for microsurgical phallic reconstruction is the radial forearm flap. The success of several different design modifications confirms the reliability of the radial and ulnar forearm flaps. Farrow et al. described their experience with the "cricket bat" concept in 1980. To the previous "cricket bat" design, we now wish to add modifications. These modifications utilize longitudinal and transverse rotations of the linear forearm tissues to create a phallus--much like the transformation of a toy robot into a truck. Deepithelialized flaps and a full-thickness skin graft coronoplasty complete glans reconstruction. The "cricket bat-transformer" flap appears to produce the most predictable results in subtotal phallic reconstructions and phallic constructions in the pediatric and transgender patient groups.     

The "sponge deformity" after tangential excision and grafting of burns

Sequential excision and grafting of burns have resulted in several new problems. We have termed one of these the "sponge deformity," i.e., a grafted area where, in multiple small areas, the bed heals underneath the graft with or without slough of the overlying graft. If the graft sloughs, a pockmark forms. If the graft does not slough, an overlying bridge forms. In our experience, this deformity is very troublesome to patients because it is difficult to wash, catches on objects, bleeds, and looks quite unsightly. Between February of 1981 and June of 1986, we treated 16 patients with this deformity. All 16 patients were treated by simple excision of the bridges and pockmark edges with a curved iris scissors. In all patients, the wounds healed well and the resultant surfaces were considerably smoother. This retrospective review of the patients suggests that the deformity usually occurs around the periphery of the excised area where the excision was shallower and when thicker grafts are used. Perhaps the bed underneath the graft epithelializes from residual epithelial elements prior to vascularization of the autograft. If this is true, it might be possible to prevent the deformity by excising the wound deeper, by applying thinner grafts, or by applying allograft or xenograft, expecting that the area will heal promptly and not require autografting.     

A comparative study of allogenic "first set" and "second set" skin rejection in mice.

In our case, where the difference between donor WHT/Ht(H--2d) and recipient C57B1/6 (H--2b) was at the H--2 locus of histocompatibility, the FSg rejection occurred between the 6th and the 10th day, while the SSg rejection was two days earlier. The morphological study emphasized that the cellular infiltrate in the FSg is predominantly lymphocytic, while in the SSg it is predominantly granulocytic. The vascularization settles on the second day postgrafting in the FSg as well as in the SSg, but is scarce and quickly destroyed in the SSg, thus being explained the scanty supply of lymphocytes in the latter graft type. The histochemical reactions demonstrated an increase of the NMPS in skin allograft from the moment of settling till the end of the rejection and a decrease of AMPS, till the complete disappearance, in the rejected graft. Little morphological differences were found between colateral draining nodes and contralateral ones during the reaction to the graft. An important feature described is the presence at the bed graft level of some cells morphologically identical to the IB encountered in great number in the TDA of lymph nodes in the allograft-bearing mice.     

"Pruning" of alloreactive CD4+ T cells using 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester prolongs skin allograft survival

Removal of alloreactive cells by either thymic deletion or deletion/anergy in the periphery is regarded as crucial to the development of tolerance. Dyes, such as CFSE, that allow monitoring of cell division suggest that in vitro proliferation could be a used as a way of "pruning" alloreactive cells while retaining a normal immune repertoire with retention of memory to previously encountered pathogens. This would overcome the problems occurring as a result of therapies that use massive depletion of T cells to allow acceptance of organ transplants or bone marrow grafts. We therefore used a skin graft model of CD4-mediated T cell rejection across a major H-2 mismatch (C57BL/6 (H-2(b)) to BALB/c (H-2(d)) mice) to evaluate whether nondividing CD4(+) T cells derived from a mixed lymphocyte culture would exhibit tolerance to a skin graft from the initial stimulator strain. We demonstrate that selective removal of dividing alloreactive CD4(+) T cells resulted in marked specific prolongation of allogeneic skin graft survival, and that the nondividing CD4(+) T cells retained a broad TCR repertoire and the ability to maintain memory. This novel way of depleting alloreactive T cells may serve as a useful strategy in combination with other mechanisms to achieve transplant tolerance.     

Hematopoietic stem cells in research and clinical applications: The "CD34 issue"

In this paper, experimental findings concerning the kinetics of hematopoietic reconstitution are compared to corresponding clinical data. Although not clearly apparent, the transplantation practice seems to confirm the basic proposals of experimental hematology concerning hematopoietic reconstitution resulting from successive waves of repopulation stemming from different subpopulations of progenitor and stem cells. One of the "first rate" parameters in clinical transplantations in hematology; i.e. the CD34+ positive cell dose, has been discussed with respect to the functional heterogeneity and variability of cell populations endowed by expression of CD34. This parameter is useful only if the relative proportion of stem and progenitor cells in the CD34+ cell population is more or less maintained in a series of patients or donors. This proportion could vary with respect to the source, pathology, treatment, processing procedure, the graft ex vivo treatment and so on. Therefore, a universal dose of CD34+ cells cannot be defined. In addition, to avoid further confusion, the CD34+ cells should not be named "stem cells" or "progenitor cells" since these denominations only concern functionally characterized cell entities.     

Radical graft polymerization of styrene sulfonate on poly(ethylene terephthalate) films for ACL applications: "grafting from" and chemical characterization

The purpose of this study is to develop a reliable method of functionalizing poly(ethylene terephthalate) with bioactive polymers to produce a "biointegrable" artificial anterior cruciate ligament. Radical graft polymerization of the sodium salt of styrene sulfonate (NaSS) onto poly(ethylene terephthalate) (PET) films was performed using the "grafting from" technique. Prior to the grafting, the surfaces of poly(ethylene terephthalate) films were activated by ozonation to generate peroxide and hydroperoxide reactive species on the PET film surfaces. The radical polymerization of NaSS was initiated by thermal decomposition of the hydroperoxides. The grafted PET surfaces were characterized by a toluidin blue colorimetric method, X-ray photoelectron spectroscopy, contact angle measurements, and atomic force microscopy. The influence of ozonation time, monomer concentration, and temperature on NaSS grafting ratios was examined. A total of 30 min of ozonation followed by grafting from a 15% NaSS solution at 70 degrees C for 90 min or more resulted in attachment of poly(NaSS) chains to the PET film surfaces.     

The use of coralline hydroxyapatite in a "biocomposite" free flap.

An animal model was developed to determine the feasibility of vascularizing a porous biomaterial and transferring it as part of a free flap to a recipient site with enhanced resistance to infection due to the maintenance of a blood supply. To that end, the experiment was divided into three stages. Stage 1 compared the soft-tissue response of Interpore-200 to Interpore-500, both continuously porous hydroxyapatite materials implanted beneath the panniculus carnosus of the male Sprague-Dawley rat. Pore size was an important factor as it influenced vascular ingrowth, with Interpore-200 vascularizing earlier (complete at 1 week) and more intensely. Interpore-200 was therefore used for the remainder of the experiment. After 1 week of tissue ingrowth, the implants were moved from the abdomen to the skull on a vascular pedicle as a "biocomposite" free flap. Stage 2 was the histologic evaluation of 15 "biocomposite" free flaps over various time intervals up to 8 months. The free flaps formed a fibrous union to the skull, while a simple nonvascularized Interpore-200 onlay graft (stage 2 control) demonstrated a bony union in three of four implants placed up to 2 months. Stage 3 confirmed the free flap's resistance to bacterial infection. A highly significant difference (p less than 0.005) in infection rates was demonstrated between the "biocomposite" and nonvascularized stage 3 controls with no Pseudomonas growth from 9 of 10 cultures of the free flaps 5 days after exposure to 10(4) Pseudomonas aeruginosa, while stage 3 controls demonstrated Pseudomonas growth in all cultures (heavy growth in 8 of 10). The "biocomposite" free flap has excellent potential to provide form and structure to wounds requiring reconstruction where bacterial contamination is a significant risk factor.     

Spontaneous renal allograft acceptance associated with "regulatory" dendritic cells and IDO

MHC-mismatched DBA/2 renal allografts are spontaneously accepted by C57BL/6 mice by poorly understood mechanisms, but both immune regulation and graft acceptance develop without exogenous immune modulation. Previous studies have shown that this model of spontaneous renal allograft acceptance is associated with TGF-beta-dependent immune regulation, suggesting a role for T regulatory cells. The current study shows that TGF-beta immune regulation develops 30 days posttransplant, but is lost by 150 days posttransplant. Despite loss of detectable TGF-beta immune regulation, renal allografts continue to function normally for >200 days posttransplantation. Because of its recently described immunoregulatory capabilities, we studied IDO expression in this model, and found that intragraft IDO gene expression progressively increases over time, and that IDO in "regulatory" dendritic cells (RDC) may contribute to regulation associated with long-term maintenance of renal allografts. Immunohistochemistry evaluation confirms the presence of both Foxp3+ T cells and IDO+ DCs in accepted renal allografts, and localization of both cell types within accepted allografts suggests the possibility of synergistic involvement in allograft acceptance. Interestingly, at the time when RDCs become detectable in spleens of allograft acceptors, approximately 30% of these mice challenged with donor-matched skin allografts accept these skin grafts, demonstrating progression to "true" tolerance. Together, these data suggest that spontaneous renal allograft acceptance evolves through a series of transient mechanisms, beginning with TGF-beta and T regulatory cells, which together may stimulate development of more robust regulation associated with RDC and IDO.     

Lymphedema of the eyelids

Lymphedema of the eyelids is a relatively rare condition whose main effect is to obstruct vision, and it is unsightly. We present 3 cases in which the edema was treated by excision and skin grafting of the eyelid, with satisfactory functional and cosmetic results. It is suggested that a split-skin graft be used for the upper eyelid and a full-thickenss graft for the lower eyelid. The lower eyelid skin can be "de-fatted" and used as a graft, but in the more severe cases we suggest a postauricular skin graft be used.     

Amphiphilic amylose-g-poly(meth)acrylate copolymers through "click" onto grafting method

Periodate oxidation and subsequent reductive amination with propargylamine was adopted for the controlled functionalization of amylose with alkyne groups, whereas ATRP polymerization was exploited to obtain end-(α)- or end-(ω)-azide functionalized poly(meth)acrylates to be used as "click" reagents in Cu(I) catalyzed azide-alkyne [3 + 2] dipolar cycloaddition. Amylose was effectively grafted with poly(n-butyl acrylate), poly(n-butyl methacrylate), poly(n-hexyl methacrylate), and poly(dimethylaminoethyl methacrylate) with this strategy. Their structure and composition were confirmed by FT-IR, NMR spectroscopies, and thermogravimetric analysis (TGA). Dynamic and static light scattering analyses, as well as TEM microscopy showed that the most amphiphilic among these hybrid graft copolymers self-assembled in water, yielding nanoparticles with ca. 30 nm diameter.     

Calibration and application of an intra-articular force transducer for the measurement of patellar tendon graft forces: an in situ evaluation.

The objective of this study was to evaluate two calibration methods for the "Arthroscopically Implantable Force Probe" (AIFP) that are potentially suitable for in vivo use: (1) a direct, experimentally based method performed by applying a tensile load directly to the graft after it is harvested but prior to implantation (the "pre-implantation" technique), and (2) an indirect method that utilizes cadaver-based analytical expressions to transform the AIFP output versus anterior shear load relationship, which may be established in vivo, to resultant graft load (the "post-implantation" technique). The AIFP outputs during anterior shear loading of the knee joint using these two calibration methods were compared directly to graft force measurements using a ligament cutting protocol and a 6 DOF load cell. The mean percent error (actual-measured)/(actual)* 100) associated with the pre-implantation calibration ranged between 85 and 175 percent, and was dependent on the knee flexion angle tested. The percent error associated with the post-implantation technique was evaluated in two load ranges: loads less than 40 N, and loads greater than 40 N. For graft force values greater than 40 N, the mean percent errors inherent to the post-implantation calibration method ranged between 20 and 29 percent, depending on the knee flexion angle tested. Below 40 N, these errors were substantially greater. Of the two calibration methods evaluated, the post-implantation approach provided a better estimate of the ACL graft force than the pre-implantation technique. However, the errors for the post-implantation approach were still high and suggested that caution should be employed when using implantable force probes for in vivo measurement of ACL graft forces.     

美味猕猴桃优良株系“实美”的砧木选择研究

美味猕猴桃优良株系“实美”的接穗嫁接于 1 0个不同种类的砧木上 ,观测其嫁接体的亲和力、生长特性、开花结果习性、抗逆性、产量、果实品质等。结果表明 :“实美”以中华猕猴桃桂海 4号为砧木 ,具有良好的亲和力 ,嫁接成活率达 1 0 0 % ,接合部位断裂率仅 5 % ;5年生植株保存率 1 0 0 % ,生长指数高达 0 .9,病死株率为 0 ;物候期相对稳定 ,萌芽率和结果枝率高 ;连续 3年的平均产量极显著地高于其它 9种砧木的产量 ;果实内含物丰富 ,品质优良。根据观测结果 ,可以认为中华猕猴桃桂海 4号是“实美”的优良砧木。     

The free tongue graft for correction of secondary deformities of the vermilion in patients with cleft lip

From January of 1985 to January of 1990, 31 patients with repaired cleft lip and secondary vermilion defects underwent 45 revisional procedures. A free tongue graft was utilized seven times in six patients (19 percent). Indications for its use were a V-shaped vermilion deficit or a "whistling" deformity associated with a sagittal vermilion deficiency and normal or insufficient lateral vermilion bulk. Of the seven free tongue grafts, none was lost. Three patients have required revisions, including repeat free tongue graft in one. Proper positioning of the graft along the free vermilion border has made color and texture match satisfactory. The free tongue graft is a simple and reliable means of transferring both vermilion bulk and surface mucosa. Introduction of the free tongue graft has eliminated the need for more cumbersome procedures, such as the Abbé flap or the tongue flap, in properly selected patients.     

Morphometric and electron microscopic study of the small intestine following total orthotopical auto- and allotransplantation]

Total orthotopic autotransplantation of the small intestine was performed in 22 dogs, allotransplantation--in 13 dogs. Changes in the wall of the graft resulted from the effect of both nonspecific (ischaemia, blockage of lymph outflow, denervation) and specific factors connected with the reaction of tissue incompatibility. Changes caused by the nonspecific factors are demonstrated as lymphostasis, edema of the wall in the graft, atrophy of the villi with epithelial flattening, hyperplasy of the intestinal gland, macrophagal and plasmocytic reaction in the mucous lamina propria, drop in villous-cryptic coefficient and reduction of the absorbing surface of the villi. At autotransplantation, structural-functional changes in the intestine undergo three stages: the stage of acute morphological changes and pronounced functional disturbances (up to 1,5 months); the stage of relative structural stabilization and gradual functional restoration (up to 6 months); the stage of atrophic-hyperplastic changes in the wall and decrease in functional activity of the intestinal graft (followed-up for 15 months). At allotransplantation, depending on the degree of cellular reaction and vascular changes, as well as on the localization of the process in the wall of the graft, a "graft versus host" reaction and a "recipient versus graft" reaction is demonstrated. Peculiar rosettes of plasmocytes have been described as a possible index of the reaction "graft versus host".     

THE POWER OF THE " A" -" NOT A" METHOD

The " A" - " Not A" method is a rating method with two categories. It is often treated as a discrimination method. Unlike forced choice procedures, the Thurstonian model for this method involves a choice criterion. In statistical tests, it is treated as a comparison of two proportions. In this paper, the power for hypothesis tests involving the monadic and replicated monadic " A" - " Not A" method is discussed. The power functions and the sample sizes needed for 80% power are given based on Thurstone's δ. Designs with equal and unequal allocations for A and A (Not A) samples are considered. The power of the method is also compared with that of four forced choice methods under the assumption that the perceptual variance is identical among methods. The comparison shows that, in general, the power for the five methods ranks from high to low: the 3-AFC, 2-AFC, " A" - " Not A", triangular and duo-trio. The comparison also shows that, based on the same number of panelists and/or the same sample size for the A and A⁻ samples for the methods, if the panelists are not too discrepant and the choice criterion in the " A" - " Not A" method is not too strict or too lax, the power of the " A" - " Not A" method is very close to that of the 2-AFC method.     

A single nucleotide polymorphism in the<Emphasis Type="Italic"> Emp3</Emphasis> gene defines the H4 minor histocompatibility antigen

Minor histocompatibility antigens (minor H antigen) elicit strong T-cell-mediated responses during both graft rejection and graft versus leukemia (GvL) among MHC-matched individuals (where MHC is major histocompatibility complex). Employing expression-cloning methodology, we have identified a cDNA clone, MI-35, encoding the immunodominant H4^b minor H antigen within the classical mouse H4 complex. The minimal antigenic epitope derived from H4^b presented on K^b class I MHC is SGIVYIHL (SYL8) and the polymorphism is due to CT nucleotide modification in p3 resulting in the change of threonine (ACT) to isoleucine (ATT). The results presented here demonstrate that amino acid variation in the allelic epitopes results in the low abundance of H4^a peptide. The differential peptide copy number resulted in an immunodominant cytotoxic T cells (CTL) response directed against H4^b while the anti-B6 response directed against H4^a was easily dominated. These results provide a molecular mechanism for the H4 minor H antigen and suggest a novel mechanism by which alloantigenic disparity caused by conservative amino acid changes can be augmented by posttranslational antigen processing events.