Reliability of cartilage digestion and FDA–EB fluorescence staining for the detection of chondrocyte viability in osteochondral grafts

The purpose of this study is to evaluate the reliability of cartilage digestion and fluorescein diacetate-ethidium bromide (FDA–EB) fluorescence staining for the detection of chondrocyte viability in osteochondral grafts. Sixteen fresh osteochondral grafts were harvested from pig knee condyles, and the articular cartilage tissue was preserved. Each cartilage graft was cut into two 70-µm thick pieces and randomly allocated to Group A or Group B. The cell viability of Group A was detected using FDA–EB fluorescence staining of the digested cartilage, and the viability of Group B was detected with FDA–EB fluorescence staining of cartilage sections. Comparisons of chondrocyte viability and correlation analyses of the two groups were performed using the paired sample t test and Pearson correlation test, respectively. No significant difference was found in the chondrocyte viability between Groups A and B (p > 0.05), and a strong correlation was observed (r = 0.70, p < 0.05). Therefore, cartilage digestion with FDA–EB fluorescence staining is a reliable method for detecting chondrocyte viability in osteochondral grafts.     

The effect of fibrin glue on skin grafts in infected sites.

Fibrin bonding of skin grafts to wounds is an essential part of the graft-adherence process. Bacteria, in concentrations greater than 10(5)/gm of tissue, are associated with graft failure. Sixty-five rats were randomly divided into three groups, dorsal split-thickness skin grafts were harvested, and the sites were inoculated with Staphylococcus aureus. After incubation, each wound was quantitatively biopsied and treated with saline, fibrin glue with aprotinin, or fibrin glue alone. We found that the addition of commercially available fibrin glue with or without the antifibrinolytic agent aprotinin is capable of restoring graft adherence to normal levels in graft sites infected with greater than 10(5) bacteria/gm of tissue. Fibrin glue may have potential for increasing skin-graft take in the clinical situation where the graft bed is infected.     

The Safety and Efficacy of Antifibrinolytic Therapy in Neonatal Cardiac Surgery

BackgroundNeonates undergoing open-heart surgery are particularly at risk of postoperative bleeding requiring blood transfusion. Aprotinin has attained high efficacy in reducing the requirement for a blood transfusion following a cardiopulmonary bypass, but is seldom studied in the neonatal age group. The aim of this study was to compare the efficacy and adverse effects of aprotinin and tranexamic acid in neonates undergoing open-heart surgery at a single centre.MethodsBetween October 2003 and March 2008, perioperative data of 552 consecutive neonatal patients undergoing open-heart surgery in Children’s Hospital Boston were reviewed. Among them, 177 did not receive antifibrinolytic therapy (Group A); 100 were treated with tranexamic acid only (Group B); and 275 patients received aprotinin with or without tranexamic acid (Group C). Except for antifibrinolytic therapy, the anaesthesiological and surgical protocols remained identical. Postoperative complications and in-hospital mortality were the primary study endpoints.ResultsBody weight and Risk Adjustment for Congenital Heart Surgery (RACHS-1) scores were statistically comparable among the three groups. No statistically significant differences were observed between the duration of hospitalization, chest tube drainage, reexploration for bleeding, and kidney function impairment. In Group C, less blood was transfused within 24 hours than in GroupB. Operative mortality was similar among the three groups.ConclusionNo further risk and kidney injury were observed in the use of aprotinin in neonatal cardiac surgery, aprotinin demonstrated a reduced requirement for blood transfusion compared with tranexamic acid. Our data provide reasonable evidence that aprotinin and tranexamic acid are safe and efficacious as antifibrinolytic modalities in neonatal patients undergoing cardiac surgery.     

Safety and efficacy of human amniotic membrane in primary pterygium surgery

Grafts made from human amniotic membrane are used to prevent recurrence of pterygium after excision. The success of the procedure can be affected by the quality of preparation and preservation of the grafts. We prospectively evaluated the safety and efficacy of cryopreserved amniotic membrane prepared at the research tissue bank of the Biotechnology Research Center in Tripoli, Libya, and used as adjunct therapy in primary pterygium excision. Twenty-six patients (15 males and 11 females) aged 21–78 years and indicated for primary pterygium excision were transplanted at the Tripoli Eye Hospital with the amniotic membrane grafts. Sixteen patients (62 %) were available for all three follow-up visits scheduled at 1, 3 and 6 months post-surgery. By the third visit, two patients (12.5 %) developed granuloma and three (18.8 %) had pterygium recurrence. The grafts were used after cryopreservation for ≤180 days or >180 days, but statistical analysis showed that the complications were not associated with the length of storage. Moreover, the high rate of complications in this study was not caused by use of cryopreserved AM. In conclusion, locally produced cryopreserved AM is safe as an adjunct therapy for treatment of primary pterygium excision.     

Human polymer-based cartilage grafts for the regeneration of articular cartilage defects

The use of autologous chondrocyte implantation (ACI) and its further development combining autologous chondrocytes with bioresorbable matrices may represent a promising new technology for cartilage regeneration in orthopaedic research. Aim of our study was to evaluate the applicability of a resorbable three-dimensional polymer of pure polyglycolic acid (PGA) for the use in human cartilage tissue engineering under autologous conditions. Adult human chondrocytes were expanded in vitro using human serum and were rearranged three-dimensionally in human fibrin and PGA. The capacity of dedifferentiated chondrocytes to re-differentiate was evaluated after two weeks of tissue culture in vitro and after subcutaneous transplantation into nude mice by propidium iodide/fluorescein diacetate (PI/FDA) staining, scanning electron microscopy (SEM), gene expression analysis of typical chondrocyte marker genes and histological staining of proteoglycans and type II collagen. PI/FDA staining and SEM documented that vital human chondrocytes are evenly distributed within the polymer-based cartilage tissue engineering graft. The induction of the typical chondrocyte marker genes including cartilage oligomeric matrix protein (COMP) and cartilage link protein after two weeks of tissue culture indicates the initiation of chondrocyte re-differentiation by three-dimensional assembly in fibrin and PGA. Histological analysis of human cartilage tissue engineering grafts after 6 weeks of subcutaneous transplantation demonstrates the development of the graft towards hyaline cartilage with formation of a cartilaginous matrix comprising type II collagen and proteoglycan. These results suggest that human polymer-based cartilage tissue engineering grafts made of human chondrocytes, human fibrin and PGA are clinically suited for the regeneration of articular cartilage defects.     

Rathke's pouch grafts in adult brain sites

Donor tissue containing Rathke's pouch (RP) with its associated mesenchyme and neural lobe was isolated from 15-day fetal rats and stereotaxically transplanted either to hypothalamic hypophysiotropic sites or to cerebral cortex of adult females for 30 days. Hosts either were intact or had been hypophysectomized 2-4 weeks prior to transplantation of Rathke's pouch. Grafts in the hypothalamus of either intact or hypophysectomized hosts were pleomorphic and large, often as wide as 1-2 mm, and occasionally larger. Grafts in the cortex of either intact or hypophysectomized hosts were nodular and occasionally projected upward in association with the meninges (cortex/meninges grafts). Certain features were characteristic of the grafts in all experimental groups, i.e., development of histotypic pars distalis with cell cords and fenestrated capillaries. In all experimental groups gonadotrophs and somatotrophs, when present, were localized at the graft margin adjacent to the connective-tissue interface; mammotrophs, when present, were distributed throughout the graft. Features specific to each experimental group also were apparent. Grafts in the hypothalamus of both intact and hypophysectomized hosts typically were encapsulated by a labyrinthine meshwork of cell processes, whereas cortex/meninges grafts directly abutted dense connective tissue or neural tissue. In hypothalamic grafts in intact hosts, moderately differentiated mammotrophs, gonadotrophs, and somatotrophs could be identified by their cytological features and immunopositivity for prolactin, luteinizing hormone, and growth hormone, respectively. In hypothalamic grafts in hypophysectomized hosts, mammotrophs were absent, and gonadotrophs and somatotrophs were poorly granulated and not abundant. Grafts in the cortex of intact hosts contained numerous, well-differentiated mammotrophs, gonadotrophs, and somatotrophs. Many of the mammotrophs in these grafts were hypertrophied, and profiles of exocytosis were common. In grafts in the cortex of hypophysectomized hosts, mammotrophs were either absent or very few, whereas gonadotrophs and somatotrophs were numerous. Gonadotrophs in these grafts were dramatically hypertrophied, although exocytosis was rare. The results indicate that development of histotypic pars distalis may occur in hypophysiotropic and non-hypophysiotropic brain sites and that the hormonal state of the host as well as implantation site modulate cytodifferentiation of specific pars distalis cell types.     

Chondrocyte transplantation for osteochondral defects with the use of suspension culture

Cartilage graft is considered to be useful in repairing chondral or osteochondral defects. One method of the cartilage graft is achieved by autologous chondrocyte transplantation following cell culture. However, chondrocytes change their phenotype during culture. We used costal chondrocytes cultured over agarose (suspension culture) as a source of graft materials. The suspension-cultured chondrocytes formed aggregate in culture. We first examined the expressions of cartilage-specific matrices of cultured chondrocytes after two weeks in culture. The chondrocytes cultured over agarose expressed more type II collagen mRNA than those cultured on plastic dishes did after two weeks in culture. Safranin O staining showed the presence of glycosaminoglycans in the chondrocyte culture over agarose, while glycosaminoglycans were not observed in the culture on plastic dishes. We then examined the changes of rat articular osteochondral defects after transplantation of suspension-cultured chondrocytes. The aggregate of suspension-cultured chondrocytes was easily picked up with forceps and transplanted in the osteochondral defects. The defects were filled with safranin O-stained hyaline cartilage tissue two weeks after chondrocyte transplantation. On the contrary, the fibrous materials, which were not stained with safranin O, were observed in the control defects. These results suggest that the suspension-cultured chondrocytes are useful for autologous cartilage grafts by preserving chondrocyte phenotype.     

Cartilage storage at 4 °C with regular culture medium replacement benefits chondrocyte viability of osteochondral grafts in vitro

Maintenance of articular cartilage allografts in culture media is a common method of tissue storage; however, the technical parameters of graft storage remain controversial. In this study, we examined the optimal temperature and culture medium exchange rate for the storage of osteochondral allografts in vitro. Cylindrical osteochondral grafts (n = 120), harvested from the talar joint surface of ten Boer goats, were randomly classified into four groups and stored under the following conditions: Group A1 was maintained at 4 °C in culture medium that was refreshed every 2 days; Group A2 was maintained at 4 °C in the same culture medium, without refreshing; Group B1, was maintained at 37 °C in culture medium that was refreshed every 2 days; Group B2, was maintained at 37 °C in the same culture medium, without refreshing. Chondrocyte viability in the grafts was determined by ethidium bromide/fluorescein diacetate staining on days 7, 21, and 35. Proteoglycan content was measured by Safranin-O staining. Group A1 exhibited the highest chondrocyte survival rates of 90.88 %, 88.31 % and 78.69 % on days 7, 21, and 35, respectively. Safranin O staining revealed no significant differences between groups on days 21 and 35. These results suggest that storage of osteochondral grafts at 4 °C with regular culture medium replacement should be highly suitable for clinical application.     

Chondrocyte suspension in fibrin glue

Autologous chondrocyte implantation has been shown to be a promising method for treatment of deep articular cartilage defects. The hyaline cartilage formed by implanted autologous chondrocytes has biomechanical properties similar to those of natural articular cartilage. Between June 2006 and September 2008 we performed Autologous chondrocyte implantation (ACI) in 50 patients and the chondrocytes were supported in fibrin glue. The cartilage biopsy samples were taken from the non-weight bearing area of the patient’s femoral condyle and the samples were transferred to the cell culture laboratory. Chondrocyte were kept in culture about 20 days. Fibrin glue was used as a three dimensional carrier for chondrocyte implantation. A 450 ml of patient’s own blood was collected prior to transplantation to produce autologous fibrinogen. Alternatively the allogenic fibrinogen was prepared from Regional Blood Center voluntary donors. Before surgery the chondrocyte suspension was mixed with fibrin glue and gel—like fibrograft was prepared. The total number of cells and the size of fibrograft depended on the defect size in the knee. Our results suggest that ACI technique with fibrin glue is a promising method for treatment of cartilage defect.     

Plasmin inhibitors prevent leukocyte accumulation and remodeling events in the postischemic microvasculature

Clinical trials revealed beneficial effects of the broad-spectrum serine protease inhibitor aprotinin on the prevention of ischemia-reperfusion (I/R) injury. The underlying mechanisms remained largely unclear. Using in vivo microscopy on the cremaster muscle of male C57BL/6 mice, aprotinin as well as inhibitors of the serine protease plasmin including tranexamic acid and ε-aminocaproic acid were found to significantly diminish I/R-elicited intravascular firm adherence and (subsequent) transmigration of neutrophils. Remodeling of collagen IV within the postischemic perivenular basement membrane was almost completely abrogated in animals treated with plasmin inhibitors or aprotinin. In separate experiments, incubation with plasmin did not directly activate neutrophils. Extravascular, but not intravascular administration of plasmin caused a dose-dependent increase in numbers of firmly adherent and transmigrated neutrophils. Blockade of mast cell activation as well as inhibition of leukotriene synthesis or antagonism of the platelet-activating-factor receptor significantly reduced plasmin-dependent neutrophil responses. In conclusion, our data suggest that extravasated plasmin(ogen) mediates neutrophil recruitment in vivo via activation of perivascular mast cells and secondary generation of lipid mediators. Aprotinin as well as the plasmin inhibitors tranexamic acid and ε-aminocaproic acid interfere with this inflammatory cascade and effectively prevent postischemic neutrophil responses as well as remodeling events within the vessel wall.     

Immunohistochemical,Ultrastructural and Functional Analysis of Axonal Regeneration through Peripheral Nerve Grafts Containing Schwann Cells Expressing BDNF,CNTF or NT3

We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN) grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs) modified to express brain-derived neurotrophic factor (BDNF), a secretable form of ciliary neurotrophic factor (CNTF), or neurotrophin-3 (NT3). Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB₄ or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.     

Comparison of organ cultured precut corneas versus surgeon-cut corneas for Descemet’s stripping automated endothelial keratoplasty

To compare precut and surgeon-cut organ cultured donor corneas for DSAEK. A total of 119 consecutive eyes treated with DSAEK were retrospectically identified. 65 grafts were cut by the surgeon (Moria, ALTK System) prior to DSAEK and 54 grafts were precut by laboratory technicians from the Danish Eye Bank (Horizon single-use system). 1 year after surgery, tomographic images were obtained with the Pentacam HR. Endothelial cell density (ECD) and best-corrected visual acuity (BCVA) was determined. Graft thickness and graft asymmetry was evaluated in the centre and 1 mm from the edge of the graft in 6 semi-meridians. 1 year after surgery, the ECD loss was similar in the two groups, averaging 25.9 ± 14 % in surgeon-cut, and 22.9 ± 17 % in precut group (p = 0.33). Mean central graft thickness was 172 ± 6 μm in surgeon-cut grafts and 182 ± 6 μm in precut grafts (p = 0.30). BCVA was similar in surgeon-cut and precut corneas; being 0.25 ± 0.02 logMAR and 0.24 ± 0.02 logMAR, respectively (p = 0.59). The graft asymmetry index was 1.48 ± 0.02 for surgeon-cut and 1.44 ± 0.02 for precut grafts. There were no significant differences in complications rate in both groups. No correlations between BCVA and central graft thickness or graft asymmetry index in both groups were observed. Organ cultured precut donor corneas are comparable with surgeon-cut grafts with respect to ECD, graft thickness and asymmetry, and postoperative complication rate.     

Ex vivo and in vivo characterization of cold preserved cartilage for cell transplantation

Due to the inconvenient and invasive nature of chondrocyte transplantation, preserved cartilage has been recognized as an alternative source of chondrocytes for implantation. However, there are major concerns, in particular, the viability and quality of the chondrocytes. This study investigated the biochemistry and molecular characterization of chondrocytes isolated from preserved cartilage for purposes of transplantation. Ex vivo characterization was accomplished by storing human cartilage at either 4 or ?80 °C in a preservation medium. Microscopic evaluation of the preserved cartilage was conducted after 1, 2, 3 and 6 weeks. The chondrocytes were isolated from the preserved cartilage and investigated for proliferation capacity and chondrogenic phenotype. Transplantation of chondrocytes from preserved cartilage into rabbit knees was performed for purposes of in vivo evaluation. The serum cartilage degradation biomarker (WF6 epitopes) was evaluated during the transplantation procedure. Human cartilage preserved for 1 week in a 10 % DMSO chondrogenic medium at 4 °C gave the highest chondrocyte viability. The isolated chondrocytes showed a high proliferative capacity and retained chondrogenic gene expression. Microscopic assessment of the implanted rabbit knees showed tissue regeneration and integration with the host cartilage. A decreased level of the serum biomarker after transplantation was evidence of in vivo repair by the implanted chondrocytes. These results suggest that cartilage preservation for 1 week in a 10 % DMSO chondrogenic medium at 4 °C can maintain proliferation capacity and the chondrogenic phenotype of human chondrocytes. These results can potentially be applied to in vivo allogeneic chondrocyte transplantation. Allogeneic chondrocytes from preserved cartilage would be expected to maintain their chondrogenic phenotype and to result in a high rate of success in transplanted grafts.     

Allograft swelling after preparation during ACL reconstruction: do we need to upsize tunnels?

The purpose of this study was to determine whether the pull-through force of soft-tissue allografts increases over time after being hydrated with saline-soaked sponges. Eighteen aseptic soft-tissue, fresh-frozen anterior tibialis allograft specimens were thawed and sized using standard sizing guides. After sizing, initial pull-through force was measured using an Instron Model 5865 machine. Grafts were randomized to soak in saline sponges for 20, 40, or 60 min. After soaking, pull-through force was again assessed. Pre- and post-soaking pull-through forces were compared using a paired t test. The effect of time on pull-through force was evaluated using an ANOVA and Tukey post hoc test. Two allografts had initial pull-through forces outside the inclusion criteria and were excluded. The average pull-through force for the remaining 16 allografts pre-soaking was 43.0 N and post-soaking was 81.7 N, for an increase of 90 % (P < 0.001). Longer hydration time in the saline soaked sponges was not correlated with higher pull-through force (P = 0.724). Pull-through force post-hydration was not related to the allograft diameter (P = 0.641). Post-hydration, 33 % of grafts that had soaked for 20 min and 40 % of grafts that had soaked for 40 or 60 min required greater than 100 N pull-through force. Our data supports the hypothesis that soft-tissue allografts swell as a result of being stored in saline-soaked sponges, resulting in greater pull-through forces during graft passage. Surgeons should bear in mind that allografts swell when stored in saline-soaked gauze and should size their tunnels accordingly.     

Cellular survival in rat vein-to-artery grafts

Microsurgical models of vein-to-artery graft surgery have been developed in rats as a means of assessing vein graft adaptation and neo-intimal hyperplasia. Neo-intimal hyperplasia in these grafts is often attributed, at least in part, to an adaptive response by venous smooth muscle cells to the increased intraluminal pressure of the arterial pressure. However, considerable evidence suggests complete or near-complete cellular replacement in these grafts. A series of experiments were undertaken in which male vein or artery grafts were placed into either allogeneic female nude rat hosts or into syngeneic WKy female hosts as a means of determining donor cell survival. Grafts were removed at postsurgery week 2 or week 6 and the fate of the donor male cells assessed by PCR amplification of the testis-determining gene Sry. The Sry gene was undetectable in 2-week male to female vein grafts. When left for 6 weeks, donor cells were detectable in vein grafts only after multiple 50-cycle PCR analyses. Minimal donor cell survival was not due to an allograft response, as donor male cells were readily detectable in WKy male to female nude rat artery-to-artery grafts. These data were not nude rat specific, as poor donor cell survival was also evidenced in syngeneic male to female vein-to-artery grafts. In conclusion, we demonstrate only marginal survival of donor cells in rat vein-to-artery grafts. Neo-intimal hyperplasia in these grafts was not a consequence of donor venous smooth muscle cell proliferation.     

Polyurethane/Gelatin Nanofibrils Neural Guidance Conduit Containing Platelet-Rich Plasma and Melatonin for Transplantation of Schwann Cells

The current study aimed to enhance the efficacy of peripheral nerve regeneration using a biodegradable porous neural guidance conduit as a carrier to transplant allogeneic Schwann cells (SCs). The conduit was prepared from polyurethane (PU) and gelatin nanofibrils (GNFs) using thermally induced phase separation technique and filled with melatonin (MLT) and platelet-rich plasma (PRP). The prepared conduit had the porosity of 87.17 ± 1.89%, the contact angle of 78.17 ± 5.30° and the ultimate tensile strength and Young’s modulus of 5.40 ± 0.98 MPa and 3.13 ± 0.65 GPa, respectively. The conduit lost about 14% of its weight after 60 days in distilled water. The produced conduit enhanced the proliferation of SCs demonstrated by a tetrazolium salt-based assay. For functional analysis, the conduit was seeded with 1.50 × 10⁴ SCs (PU/GNFs/PRP/MLT/SCs) and implanted into a 10-mm sciatic nerve defect of Wistar rat. Three control groups were used: (1) PU/GNFs/SCs, (2) PU/GNFs/PRP/SCs, and (3) Autograft. The results of sciatic functional index, hot plate latency, compound muscle action potential amplitude and latency, weight-loss percentage of wet gastrocnemius muscle and histopathological examination using hematoxylin–eosin and Luxol fast blue staining, demonstrated that using the PU/GNFs/PRP/MLT conduit to transplant SCs to the sciatic nerve defect resulted in a higher regenerative outcome than the PU/GNFs and PU/GNFs/PRP conduits.     

The effect of femoral tunnel placement on ACL graft orientation and length during in vivo knee flexion

Anatomically placed grafts are believed to more closely restore the function of the ACL. This study measured the effect of femoral tunnel placement on graft orientation and length during weight-bearing flexion. Both knees of twelve patients where the graft was placed near the anteroproximal border of the ACL and ten where the graft was placed near the center of the ACL were imaged using MR. These images were used to create 3D models of the reconstructed and intact contralateral knees, including the attachment sites of the native ACL and graft. Next, patients were imaged using biplanar fluoroscopy while performing a quasi-static lunge. The models were registered to the fluoroscopic images to reproduce in vivo knee motion. From the relative motion of the attachment sites on the models, the length and orientation of the graft and native ACL were measured. Grafts placed anteroproximally on the femur were longer and more vertical than the native ACL in both the sagittal and coronal planes, while anatomically placed grafts more closely mimicked ACL motion. In full extension, the grafts placed anteroproximally were 12.3±5.2° (mean and 95%CI) more vertical than the native ACL in the sagittal plane, whereas the grafts placed anatomically were 2.9±3.7° less vertical. Grafts placed anteroproximally were up to 6±2 mm longer than the native ACL, while the anatomically placed grafts were a maximum of 2±2 mm longer. In conclusion, grafts placed anatomically more closely restored native ACL length and orientation. As a result, anatomic grafts are more likely to restore intact knee kinematics.     

Effects of rhBMP-2 and osteopromotive membranes on experimental bone grafting.

A problem with using autogeneic bone for onlay grafting is its degree of incorporation and volumetric persistence. This study explored whether improved graft volumetric maintenance can be achieved by recombinant human bone morphogenetic protein-2 (rhBMP-2) and whether a combination of rhBMP-2 and osteopromotive membranes is advantageous in this respect. Unicortico-cancellous bone grafts were positioned below the temporal muscle bilaterally in 48 adult Lewis rats. The recipient bone was ground, and the grafted area was randomly treated according to one of eight different alternatives. Two doses of rhBMP-2 (4 microg/80 microl or 32 microg/80 microl buffer) in a collagenous carrier were tested, with or without coverage with expanded polytetrafluoroethylene membranes. As controls, membrane and carrier, alone or in combination, and onlay grafts only were used. The results were evaluated after 4 and 20 weeks by routine histologic examination and immunohistochemical labeling for various bone and cartilage matrix proteins. After 4 weeks, rhBMP-2-treated grafts showed complete integration, whereas for controls, only the membrane-alone group attained incorporation after 20 weeks. The combined treatment with high-dose rhBMP-2 and membrane demonstrated, as compared with the remaining rhBMP-2 groups, pronounced bone formation and less graft resorption, resulting in maintained or increased graft size. This was observed after 4 weeks, and the result remained at 20 weeks. Treatment with high-dose rhBMP-2 without membrane placement, in contrast, resulted in extensive resorption and graft size reduction at 20 weeks. Control groups showed less success in graft size persistence. It was concluded that a combination of high-dose rhBMP-2 and osteopromotive membranes had a synergistic effect, leading to rapid, complete graft integration and size maintenance. In the absence of a membrane, the protein seemed to accelerate the remodeling of the graft.     

Tubular silk scaffolds for small diameter vascular grafts

Vascular surgeries such as coronary artery bypass require small diameter vascular grafts with properties that are not available at this time. Approaches using synthetic biomaterials have been not completely successful in producing non-thrombogenic grafts with inner diameters less than 6 mm, and there is a need for new biomaterials and graft designs. We propose silk fibroin as a microvascular graft material and describe tubular silk scaffolds that demonstrate improved properties over existing vascular graft materials. Silk tubes produced using an aqueous gel spinning technique were first assessed in vitro in terms of thrombogenicity (thrombin and fibrinogen adsorption, platelet adhesion) and vascular cell responses (endothelial and smooth muscle cell attachment and proliferation) in comparison with polytetrafluoroethylene (PTFE), a synthetic material most frequently used for vascular grafts. Silk tubes were then implanted into the abdominal aortas of Sprague-Dawley rats. At time points of 2 weeks and 4 weeks post implantation, tissue outcomes were assessed through gross observation (acute thrombosis, patency) and histological staining (H&E, Factor VIII, smooth muscle actin). Over the 4-week time period, we observed graft patency and endothelial cell lining of the lumen surfaces. These results demonstrate the feasibility of using silk fibroin as a vascular graft material and some advantages of silk tubes over the currently used synthetic grafts.