Development of CaCO3 microsphere-based composite hydrogel for dual delivery of growth factor and Ca to enhance bone regeneration.

Injectable scaffolds have attracted much attention because of their minimum surgical invasiveness. However, limited osteogenic induction property and low mechanical properties hampered their application in bone tissue engineering. CaCO3 microspheres, which possess osteoinductivity, rough surfaces and specific binding sites for BMP-2, were first fabricated; after BMP-2 uploading, microspheres were further entrapped in fibrin-glue hydrogel. CaCO3 microspheres were co-functionalized with casein and heparin. To obtain a high encapsulation of heparin and thus BMP-2 uploading, along with controlled release and simultaneous maintenance of the presence of vaterite which had osteogenic induction property, fabrication parameters were optimized and microspheres were characterized using XRD, FITR and SEM. The formed CaCO3 had a microsphere morphology of ∼1 µm. Both vaterite and calcite phases were present and the relative amount of calcite phase increased with the amount of heparin. Sample 25 mM_4-1Hep with the highest loading amount of heparin was selected as carrier for BMP-2 and BMP-2 loaded CaCO3 microspheres were further entrapped in fibrin-glue hydrogel (FC-B). For the as-prepared composite hydrogel, mechanical properties were characterized and the presence of CaCO3 significantly elevated the tensile strength; controlled release of BMP-2 was sustained until day 21. Based on ALP activity, alizarin red staining and RT-PCR, in vitro osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was found to be significantly enhanced under induction of FC-B. Rabbit tibia bone defect model was applied to evaluate its in vivo performance. After implantation for 4 weeks, presence of composite hydrogel was observed in defects. After 8 weeks, bone defects of FC-B group were nearly completely healed. Using the fact that autologous scaffolds can be derived based on fibrin-glue hydrogel, the well-designed BMP-2 loaded fibrin-glue composite hydrogel demonstrated good potential in bone tissue engineering.

Comparison of self-made cryopreservative fibrin glue and commercial fibrin glue kit in pterygium surgery: 1-year follow-up.

PURPOSE: To assess long-term efficacy and safety of self-made cryopreservative fibrin glue (SMC) applied in pterygium surgery. METHODS: Prospective, comparative, interventional case series. Forty eyes of 40 patients with nasal primary pterygium, 24 male and 16 female, were enrolled. The patients were assigned to two groups and each contained 12 male and eight female based on the pterygium area encroaching onto the cornea. In one group, the conjunctival autograft was attached to the sclera with SMC stored for 2 months, and in the other group, commercial fibrin glue kit (CK) was applied after the pterygium was removed. All the patients were followed up postoperatively on days 1, 3, 7 and 14 then at months 1, 3, 6, 12. The main outcome measures included operating time, postoperative discomfort, recurrence rate and complications. RESULTS: There were no significant differences in surgery time (p = 0.713) and postoperative discomfort (day 1, 3, 7; p = 0.747, p = 0.766, p = 0.983, respectively) between the two groups. By the end of 1-year follow-up, the recurrence rate was 0% in the SMC group and 5% in the CK group (p = 1.000). There were no infections and severe visual acuity (VA) threatening complications in either group. CONCLUSION: Self-made cryopreservative fibrin glue (SMC) is as effective as standard CK for autograft fixation in pterygium surgery and it also has good safety after long-term follow-up. For its convenience and low cost, this new methods should be popularized, especially in underdeveloped area.

Enhancement of withstanding pressure of fibrin sealant by modified mixing ratio of fibrin sealant components for skull base reconstruction: technical note.

A method to enhance the withstanding pressure of fibrin sealant in gasket-seal closure to prevent cerebrospinal fluid (CSF) leakage after extended transsphenoidal surgery (ETSS) was investigated by adjusting the mixing ratio of the components. A plastic chamber (200 ml) was constructed with a lid made of hydroxyapatite with a hole 10 mm in diameter. The chamber could be pressurized via an opening in the side wall. The hole in the hydroxyapatite lid was covered with a Gore-Tex sheet, 15 mm in diameter. The margin of the sheet was free. Solutions A (fibrinogen 80 mg/ml) and B (thrombin 250 units/ml) of fibrin sealant were mixed in volume ratios of 1:1, 2:1, and 5:1, and applied to the Gore-Tex sheet, then water was introduced to cover the fibrin sealant. The pressure was measured at which air leakage occurred from the side of the Gore-Tex sheet. The pressure values for A/B ratios of 1:1, 2:1, and 5:1 were 117 ± 23.8 mmH(2)O (mean ± standard error) (n = 5), 234 ± 38.8 mmH(2)O (n = 5), and 345 ± 36.4 mmH(2)O (n = 5), respectively, in the acute phase (5 minutes after application of fibrin sealant). Pressures were increased after 24 hours, and that for 5:1 was the highest (373 ± 40.4 mmH(2)O, n = 5). The use of devices such as syringes specially designed to mix solutions A and B in the ratio of 5:1 can easily enhance the preventive effect of fibrin sealant against CSF leakage in ETSS.

New method to prepare autologous fibrin glue on demand.

Fibrin-based sealants are commonly employed to arrest bleeding after surgery. Usually, fibrinogen obtained from pooled human plasma is used to prepare sealants, with attendant risk of blood-borne infections. Availability of autologous fibrinogen would eliminate this risk. To prepare autologous fibrin sealant, fibrinogen was precipitated from human plasma using protamine. Under optimal conditions (10-mg/mL protamine and 22 degrees C), 96 +/- 4% of clottable fibrinogen was recovered by a simple and inexpensive technique. Nearly 50% of the plasma factor XIII was also recovered with the fibrinogen. Using bovine thrombin, the fibrinogen was clotted (1) in a specially designed mold to measure tensile strength and (2) in a lap joint between 2 aortic vessel strips to measure adhesion strength. Tensile and adhesion strengths increased with increasing fibrinogen concentration, and they were increased by the addition of calcium chloride. The addition of aprotinin and -aminocaproic acid to the fibrinogen concentrate before clotting had no effect on the mechanical properties of the clots. After adding thrombin to sealant containing 15-mg/mL fibrinogen, maximum tensile strength was achieved in 1-5 min, and maximum adhesion strength was reached in 5-15 min. For the sealant with 30-60-mg/mL fibrinogen and added calcium, the tensile strength was equivalent to that of the commercial fibrin sealant Tisseel. The adhesion strength of sealant with 30-60-mg/mL fibrinogen exceeded the adhesive strength of Tisseel under identical conditions. Autologous fibrin sealant is an attractive alternative to commercial sealants. It can be readily prepared from 5-mL plasma or more and exhibits mechanical properties equivalent to those of the leading commercial sealant.

Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution.

Platelet-rich fibrin (PRF) belongs to a new generation of platelet concentrates geared to simplified preparation without biochemical blood handling. In this initial article, we describe the conceptual and technical evolution from fibrin glues to platelet concentrates. This retrospective analysis is necessary for the understanding of fibrin technologies and the evaluation of the biochemical properties of 3 generations of surgical additives, respectively fibrin adhesives, concentrated platelet-rich plasma (cPRP) and PRF. Indeed, the 3-dimensional fibrin architecture is deeply dependent on artificial clinical polymerization processes, such as massive bovine thrombin addition. Currently, the slow polymerization during PRF preparation seems to generate a fibrin network very similar to the natural one. Such a network leads to a more efficient cell migration and proliferation and thus cicatrization.

Evaluation of a fibrin-based skin substitute prepared in a defined keratinocyte medium.

The purpose of this study was to evaluate the influence of fibrin glue and aprotinin on the growth of adult human skin keratinocytes in defined serum-free conditions. The keratinocytes were cultured on cell culture plastics and on a fibrin matrix prepared from fibrin glue. The cell growth was measured by MTT assay, while the growth of clonogenic keratinocytes was evaluated by colony assay and expressed as colony-forming efficiency (CFE). The clonogenic potential of keratinocytes released from subconfluent and confluent cultures grown on fibrin glue was also studied by the colony assay. In comparison to a plastic culture surface the fibrin glue had significantly (P<0.05) increased the clonogenic potential of keratinocytes, as well as enhanced their growth. Keratinocytes released from subconfluent cultures grown on fibrin glue attained a significantly (P<0.05) higher percentage of clonogenic cells than their confluent parallels. At 75, 150, 300 and 450 KIU/ml aprotinin did not influence the growth of keratinocytes (P>0.2). A fibrin-based skin substitute produced in the defined keratinocyte medium could be safely used to treat a number of skin defects.

Autologous fibrin glue with growth factors in reconstructive maxillofacial surgery.

The aim of this paper was to describe a method for the preparation of autologous fibrin glue with platelet growth factors and to report its use with particulate cancellous bone in reconstructive maxillofacial surgery. The fibrin glue is a two-component glue, where the one component is a concentrated fibrinogen solution with platelet growth factors and the other component is a thrombin solution. Both components were produced from the patients own blood, thus making the glue entirely autologous. The glue was prepared from platelet rich plasma separated from 200 ml of the patient's blood prior to the operation. The fibrinogen in the glue was precipitated from the platelet rich plasma by ethanol precipitation at low temperature and separated together with the platelets by centrifugation. Raising the temperature to 37 degrees C redissolved the precipitate. The thrombin solution in the glue was produced from prothrombin precipitated from 10 ml of the platelet rich plasma by lowering the pH and the ionic strength. The precipitate was separated by centrifugation and dissolved in a calcium ion solution. Increasing the pH to neutral value induced activation to thrombin. Preparation of the fibrin glue was performed in the blood bank within 60 to 90 min with the use of standard equipment. The outcome from 200 ml of blood was approximately 8 ml of fibrin glue: 6 ml fibrinogen to be coagulated with 2 ml of thrombin. The glue had a fibrinogen concentration of approximately 12 times the value in platelet rich plasma and the concentration of growth factors was approximately eight times the value in platelet rich plasma. We have used this glue successfully with particulate bone grafts for reconstructive purposes within the oral and maxillofacial field. It might as well be applied to other surgical areas. Whenever larger amount of the glue will be needed, a whole unit of blood may be taken from the patient, and the red cells re-transfused to the patient during or after the operation.

Technical manual for manufacturing autologous fibrin tissue adhesive.

PURPOSE: The aim of this article is to provide a concise and simple technical manual for manufacturing autologous fibrin tissue adhesive derived from the precipitation of fibrinogen using a combination of ethanol and freezing for surgery. METHODS: All materials and equipment needed to manufacture ethanol-based autologous fibrin tissue adhesive are listed. In addition, step-by-step instructions are provided to allow for easy and rapid fibrin adhesive production. RESULTS: Ethanol-based autologous fibrin tissue adhesive can be manufactured in under 60 minutes. Furthermore, at our institution the startup cost for manufacturing ethanol-based autologous fibrin tissue adhesive was under $2,500.00. CONCLUSION: Ethanol-based autologous fibrin tissue adhesive is a safe, reliable, and easily manufactured autologous fibrin tissue adhesive that can be made by a trained technician in any blood bank, pharmacy, or surgical laboratory.

Use of fibrin sealant to reduce bleeding after needle liver biopsy in an anticoagulated canine model: work in progress.

PURPOSE: Needle biopsy of the liver is a common diagnostic procedure. Although relatively safe, bleeding remains a potential complication and may occur more frequently in patients with coagulopathy. The purpose of this study was to evaluate the utility of a fibrin sealant in preventing bleeding after a 15-gauge needle biopsy of the liver in a canine model heparinized to simulate coagulopathy. MATERIALS AND METHODS: Fibrin sealant was delivered to biopsy tract sites in eight dogs anticoagulated with heparin (activated clotting time 387 seconds +/- 94) using the same sheath system that was employed to obtain the biopsy specimen. RESULTS: The results demonstrated complete hemostasis in the sealant-plugged tracts as compared to controls. Continuous bleeding was observed in none of the fibrin sealant-treated sites, compared with all of the control biopsy sites (P = .0078). CONCLUSION: These results demonstrate the high degree of efficacy of fibrin sealant delivered through a sheath system in plugging liver biopsy tracts and eliminating bleeding in a severely coagulopathic animal model. This indicates that fibrin sealant use in cutting needle biopsies can reduce major and minor complications associated with the procedure.

Fibrin sealant: an evaluation of methods of production and the role of the blood bank.

Blood banks can prepare fibrin sealant by several methods. Allogeneic components allow for banking of the fibrinogen concentrate for immediate use. Autologous components eliminate the risk of transfusion-transmitted disease to the recipient, but not necessarily to the preparer. Ethanol and ammonium sulfate precipitation of fibrinogen concentrate allow use of autologous blood and fast preparation (less than 90 minutes). Cryoprecipitation from liquid plasma is adequate, conserving fresh frozen plasma. Cryoprecipitation by the "freeze-thaw" method has been reported to have the highest fibrinogen yield (7840 mg/dL +/- 1800 mg/dL), whereas ammonium sulfate precipitation has been reported to have the highest bonding strength (41 g/cm2 10 minutes after thrombin addition). Cost, storage, preparation time, and transfusion-transmitted disease all play a role in choice of method.

Platelet gel: an autologous alternative to fibrin glue with applications in oral and maxillofacial surgery.

The preparation and use of platelet gel, an autologous formulation of fibrin glue, are described. The unique features of this biologic sealant are that it is derived from autologous blood collected in the immediate preoperative period by the anesthesiologist, it contains a high concentration of platelets, and it can be used in patients who are not candidates for blood bank donation. Platelet gel has been used successfully in the area of reconstructive oral and maxillofacial surgery in conjunction with ablative surgery of the maxillofacial region, mandibular reconstruction, surgical repair of alveolar clefts and associated oral-antral/ oral-nasal fistulas, and adjunctive procedures related to the placement of osseointegrated implants.

Effect of Tisseel on healing after periodontal flap surgery.

The purpose of this investigation was to evaluate the effect on healing of fast and slow absorbable Tisseel in combination with periodontal flap surgery. Mucoperiosteal flaps were raised on the buccal aspect of maxillary premolars and mandibular premolars and first molars in 4 beagle dogs. The underlying buccal, interproximal and inter-radicular bone was then removed to a level of approximately 5 mm apically to the original bone crest and half way into the interdental spaces and bifurcations. The exposed root surfaces were curetted in order to remove the periodontal ligament tissue, and a notch was made in the root surface at the base of the defects. On the control teeth, the flaps were sutured immediately after creation of the defects, while on the test teeth, a layer of fast (group I) or slow (group II) absorbable Tisseel was applied between the curetted roots and the subsurface of the flaps prior to suturing. Postoperatively, the teeth were brushed 2 x weekly. The dogs were sacrificed after 4 months. Histological analysis revealed that the amounts of new attachment and bone regrowth were similar in the test and control groups, although the results tended to be most favorable for the group of teeth treated with fast absorbable Tisseel (Group I).

A simple method of preparation of autologous fibrin glue by means of ethanol.

We describe herein a new system for the preparation of autologous fibrin glue by means of ethanol. The system produces a good yield of fibrin glue with a high concentration of fibrinogen in a short period of time, making the glue an efficient hemostatic agent and surgical sealant, and autologous fibrin glue has the obvious advantages of safety from transmission of viral agents and from immunologic reactions.

Autologous fibrin glue--preparation and clinical use in thoracic surgery.

Autologous fibrin glue was used in 20 patients undergoing lung resection to reduce pulmonary air leaks and improve hemostasis. The fibrinogen in the glue was prepared by ethanol precipitation of plasma separated from 88 ml of the patient's blood. The mean volume of fibrinogen concentrate +/- SD was 4.9 +/- 0.5 ml with a fibrinogen concentration of 28 +/- 5 mg/ml. The yield obtained by the separation was 81% +/- 9%. One part of fibrinogen concentrate was converted to solid fibrin by means of 0.3 parts of thrombin solution. The outcome was 6.4 ml of two-component fibrin glue. The preparation was performed in a closed system to ensure sterility, and was completed within 90 min. Pulmonary air leak decreased following sealing of the resection lines with autologous fibrin glue and the hemostasis was effective. No adverse effects were observed, and all cultures from the glue were negative. Autologous fibrin glue has the obvious advantages of safety from transmission of viral diseases and from immunological reactions. In summary, we report a new technique for preparing autologous fibrin glue with a high concentration of fibrinogen making it a safe and effective sealant of pulmonary air leak and hemostatic agent in thoracic surgery.

Four years' experience with fibrin sealant in thoracic and cardiovascular surgery.

A single-donor fibrin sealant system was used in 689 thoracic and cardiovascular surgical procedures over the 4-year period between April 1, 1985, and March 31, 1989. An excellent overall success rate (646/689, 94% effective) was achieved with specific applications, including reduction of leakage of air (29/33, 88% effective), blood (595/634, 94% effective), and fluid (14/14, 100% effective), as well as positioning of anatomical structures such as coronary bypass grafts (8/8, 100% effective). Application methods included use of spray bottles (477/497, 96% effective), syringes (165/186, 89% effective), and a Silastic cannula through the flexible fiber-optic bronchoscope (4/6, 67% effective). The system was used in a wide variety of cardiac, pulmonary, esophageal, and vascular procedures to seal staple lines, suture lines, anastomoses, conduits, fistulas, and raw surfaces. No complications with this single-donor system secondary to blood-borne disease have been documented. Overall infection occurred at a nominal rate (16/689, 2%). Thus, fibrin sealant has been a useful tool to control the leakage of air, blood, and fluid during a wide variety of thoracic and cardiovascular procedures and may be of benefit to other surgeons.

The use of fibrin glue in intracranial procedures: preliminary results.

Intracranial procedures always have the potential of cerebrospinal fluid (CSF) leakage postoperatively. Sealing all routes of CSF drainage to the outside of the intracranial contents is essential. This is usually achieved with muscle and fat plugs, homograft dura, fascia, and suture. The use of a fibrin glue might affect and lessen the likelihood of a CSF leak. Eight clinical cases of various intracranial procedures using a simple, two-part patient autologous cryoprecipitate fibrinogen and bovine thrombin glue are described. Preliminary results of up to 1 year show no CSF leakage nor adverse reactions to the fibrin glue. The production method and material characteristics are briefly compared with other currently described autologous fibrin glue formulations. This version is similar in strength to other formulations, yet is simpler and more convenient to produce. The use of this autologous fibrin glue appears to provide an adjunct to commonly employed packing techniques in a convenient and effective manner. With more experience, fibrin glue might become an even more important tool in intracranial procedures.