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Tissue Engineering/ Biomaterials

Hemostasis and Bone Regeneration Using Chitosan/Gelatin-BCP Bi-layer Composite Material

Padalhin, Andrew R.*; Lee, Byong-Taek

Author Information
doi: 10.1097/MAT.0000000000000850

Abstract

Blood is a specialized connective tissue that not only serves to maintain the form of the body and organs, but also transport elements for normal physiologic function. Together with the various network of vessels, blood is the main media for distributing nutrients, oxygen and for removal of toxins and metabolic wastes. Severance of blood vessels leads to leakage of this vital loose connective tissue. Hemorrhage could lead to serious conditions or even death. Advancements in the medical field have resulted into development of copious amounts of biomaterials, drug delivery systems, and tissue engineering scaffolds for tissue regeneration and disease treatments. Tissue damage due to injury or surgical procedure often leads to release of bodily fluids and blood. Under these circumstances, hemostasis should be achieved properly to prevent critical blood loss.1–7 Preventing excessive bleeding is quite manageable when vascularized pliable tissue is involved however it presents a very challenging task when blood is surging from a bone defect. This is because hemostasis is primarily achieved through the formation of a stable clot brought about by platelet activating factors or occlusion of the collapsible source such as blood vessels. Hemostasis can be attained through the application of pressure with the use of absorbent material, closing off with a hemostat clamp, cauterizing, or application of chemical agents for blood coagulation.2–5 It is quite difficult to cease bleeding in bone because it is a very rigid tissue that maintains its form even upon application of pressure. Thus, bleeding continues until an actual physical barrier is formed to counter act the blood flow. This is accomplished using either ample amounts of cotton gauze or via smearing of bone wax.8 However, several findings indicate that using bone wax as a hemostatic agent could induce undesirable effects that could eventually affect bone regeneration.9–11

A number of natural and synthetic polymers have already been used to create hemostatic agents for clinical application: collagen, cellulose, chitosan, and gelatin to name a few.3–6 Among these polymers, gelatin and chitosan have been chosen for this study. Chitosan is a polysaccharide generated from treatment of crustacean shells. This natural polymer has been used in numerous applications throughout the food industry and biomedical field due to its versatility, biodegradability, and biocompatibility.12,13 Chitosan has also been proven to have antibacterial activity against several bacterial species depending on the chitosan solution properties.14–16

Gelatin is a natural polymer derived from denatured collagen which mainly makes it a suitable component for biomaterials and tissue scaffolds. Numerous applications have already been employed using gelatin as a main scaffold component17,18 and as a composite constituent for drug delivery system.19 Gelatin is generally used to produce tissue scaffolds conducive for cell attachment and tissue development. It is one of the most common components used in hemostatic materials because like chitosan it is capable of absorbing substantial amounts of fluid and swell.20,21 Calcium phosphate powders such as hydroxyapatite and biphasic calcium phosphate (BCP) has been proven and used in countless applications for bone and dental regeneration and reconstruction due to osteoconductive and biocompatibility.21–23

In this study, a double-layered composite hemostatic material was fabricated by combining chitosan and gelatin. The material consists of a sheet of freeze-dried chitosan hydrogel and a thin layer of electrospun gelatin-BCP fibers designed as a degradable and absorbent alternative to bone wax. The design allows the absorption of blood fluid by the hydrogel which then assists in the filtration of major particulate components of blood along the fibrous electrospun layer to create and maintain a stable clot.

Methods

Fabrication of Chitosan/Gelatin-BCP Composite Material

Materials for fabricating the composite hemostat: gelatin (type A, from porcine skin) and chitosan (de-acetylated shells from shrimp) were purchased from Sigma-Aldrich, MO. While BCP was synthesized using a microwave-assisted process.24 Solvents used to dissolve the powder components: 2,2,2-trifluoroethanol (TFE, 99.0%) and glacial acetic acid were procured from Fluka, Buchs, Switzerland. Dimethylsulfoxide (99.0%) was obtained from Samchun Pure Chemical Co., Ltd. (Pyeongtaek City, South Korea). Minimum essential medium (MEM) was obtained from HyClone (Logan, UT), fetal bovine serum (FBS), penicillin/streptomycin, mice preosteoblast (MC3T3-E1) cells were obtained from the Korean Cell Bank (Seoul, South Korea). All chemicals and solvents were of analytical reagent grade.

The hydrogel was prepared by dissolving 2% weight/volume (w/v) of chitosan in 1% acetic acid at elevated temperature (40°C). The resulting solution was filtered to remove large impurities. Then 2 ml of the viscous chitosan solution was poured into each well of a 24-well plate. The plate was then transferred to 4 °C for 8 hours afterward freezing was done at –20°C for 8 hours. To form the pores and solidify the structure of the hydrogel, frozen chitosan was then freeze-dried for 48 hours to eject the ice crystals from the molded material.

The gelatin-BCP solution was prepared by dissolving 15% w/v gelatin dissolved in 2, 2, 2-trifluoroethanol (TFE, 99.0%) at elevated temperature (40°C) of with continuous stirring until a homogenous solution is achieved. Then 50% w/w BCP powder, synthesized using a microwave-assisted process was added into the gelatin solution. The mixture was stirred for another 8 hours until a homogenous solution was achieved. The gelatin-BCP solution was then loaded in a 10 ml Luer-lock syringe equipped with a 21 gauge needle and placed in an electrospinning setup. To fabricate the bi-layered composite, the freeze-dried chitosan hydrogels were lightly attached to a rotating metal collecting drum then electrospinning of the gelatin-BCP solution was initiated at 15 kV, flow rate of 1 ml/h, 10 cm collector-tip distance. After creating the composite, each piece of sample was carefully removed from the collector drum. To ensure the stability of the fibrous layer upon blood contact, crosslinking of the gelatin-BCP was done using EDC-NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride - N-hydroxysuccinimide) solution as per reference (25). Excess crosslinking agent was removed from the samples with repeated washing using phosphate buffered solution followed by distilled water. The samples were then freeze-dried for 24 hours before use for material characterization and biocompatibility testing.

Characterization of Chitosan/Gelatin-BCP Composite Hemostat

Cross sections of the chitosan hydrogel and the surface morphology of the gelatin-BCP mat was examined for pore and fiber morphology respectively using a scanning electron microscope (SEM, JEOL, JSM-6701F, Japan). After fabrication, dried samples were mounted on an SEM mount using adhesive carbon tape and was coated with platinum (Cressington 108 Auto). Energy dispersive spectrometry (EDS) profile as taken on the gelatin-BCP fibers to confirm presence of the powder within the formed fibers.

Cell Adhesion and Proliferation

Proliferation and adhesion on the composite bi-layered hemostat were observed using preosteoblast (M3CT3-E1) commercial cell line. A sample of the composite chitosan/gelatin-BCP hemostat measuring 10 mm in diameter was sterilized using ultraviolet (UV) irradiation and conditioned with MEM media containing 10% FBS and 5% penicillin streptomycin (PS) for 5 minutes after which a density of 2 × 104 cells/ml of M3CT3-E1 preosteoblast cells was seeded unto the samples. Media was replaced every other day within the 1 week culture period. Cell proliferation on the seeded composite chitosan/gelatin-BCP hemostat was measured from 1, 3, 5, and 7 days after seeding by conducting MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, comparing the data with that of the electrospun gelatin mat as a control. Briefly, 100 µl of MTT solution was added in each well; then the samples were reincubated for 4 hours. Media was then removed from each well and the remaining formazan salt was dissolved by adding 1 ml of dimethylsulfoxide and shaking for 1 hour. The resulting solution was transferred into a 98 well plate, and then the absorbance was read at 595 nm using a microplate reader (Infinite F50, Tecan, Austria). A standard curve relating the absorbance values with a corresponding cell count was established. The standard curve was then used to determine the estimated cell count for each observation period. The data were then graphed as fold change of cell population over time. Cell adhesion was observed by staining paraformaldehyde (4%) fixed samples with fluorescein isothiocyanate (FITC, sigma) and 4’,6’-diamidino-2-phenylindole. Micrographs of each sample were taken using confocal microscope (Olympus FV 10i, Japan).

Ex Vivo Blood Absorption

To test the absorption capability of the composite bi-layer material, 10 mm diameter by 3 mm thick samples were prepared and sterilized using UV light. Anticoagulated blood was extracted from sacrificed rats by collecting blood into a 10 ml syringe containing 1 ml of acid citrate dextrose. Then 100 ml of anticoagulated blood was dropped on electrospun surface of the 9 samples. Three samples were then removed from the setup after 1, 3, and 5 minutes. These samples were then gently rinsed in phosphate buffered saline to remove nonadherent blood cells. Samples were then placed in 1 ml distilled water to lyse the attached red blood cells. The solutions were then centrifuged at 2500 rpm for 5 minutes and the supernatant was then read at 540 nm to quantify the amount of free hemoglobin.

In Vivo Hemostatic Activity and Implantation Tests

To determine hemostatic capability of the bi-layer chitosan/gelatin-BCP composite and its effect on bone regeneration, the sample material was tested as a topical hemostatic agent and a degradable hemostat. Animal experiments were conducted based on the guidelines approved by the Animal Care Center, Soonchunhyang University, Chungcheongnam-do, South Korea. Before conducting the experiment, 12 male Sprague-Dawley weighing 300–350 g were acclimated in individual cages and provided with adequate food and water.

The animals were anesthetized using 2% isoflurane gas vaporized using a Isoflurane funnel-fill vaporizer (Harvard Apparatus, 34-1040SV) attached to an oxygen tank with appropriate regulator valves. After the animal loses its righting reflex, cranial region was shaved, cleaned with 70% ethanol and disinfected with povidone solution. Using sterile surgical blade, an incision was created to expose the underlying parietal plates of the skull. Two noncritical sized, full-thickness circular defects measuring 3 mm in diameter were created on both sides using a sterile trephine drill at a rotating speed of 1,500 rpm or less with constant sterile saline irrigation to prevent overheating. The drilling procedure was conducted with gentle pressure taking note of the subtle curvature of the bone surface to prevent over penetration. Bleeding was achieved once the bone plate was completely removed from the defect area, thus creating a hemorrhagic bone model. For the control defect, bleeding was allowed to proceed on the right-side defect until it gradually formed a stable clot and achieved natural hemostasis.

Hemostatic activity was tested by pressing down a 10 mm diameter sample on the left side defect right after achieving bleeding upon removal of the bone piece from the defect. The sample was removed after bleeding was successfully stopped. To determine the effect of the bi-layered chitosan/gelatin-BCP hemostat on bone tissue development, a sample measuring 3 mm diameter was also implanted as a degradable hemostat after achieving sufficient hemostasis into similar sized defects on left side while a control defect was maintained on the right side. Hemostasis using a bone wax substitute (BWS) (Ostene, Ceremed, CA) was also tested for comparison. Left side defects were created to which BWS was applied to cease bone bleeding.

Incisions were then closed with 0–5 Vicryl suture after application of the samples and were subsequently sterilized with povidone iodine solution. All rats were subcutaneously injected with a prophylactic (Baytril, Bayer) at 10 mg/kg. Six rats were utilized for topical hemostatic testing, degradable hemostat testing and BWS application, adding up to a total of 18 rats with each rat having its right side defect as a control reference.

The animals were then allowed to recover with ad libitum food and water after which two rats from each treatment were sacrificed after 1 and 3 weeks. Extracted skull plates were fixed using 10% formalin and were subjected to Micro CT analysis before tissue embedding. Tissue samples were scanned with a micro CT scanner (Skyscan 1076, Belgium), and resulting raw data were then reconstructed using Nrecon (Bruker, Belgium). The reconstructed data were analyzed using CTan (Bruker, Belgium) using a uniform volume of interest based on appropriate threshold values to eliminate signal noise. Three-dimensional reconstruction of the analyzed data was rendered using CTVox (Bruker, Belgium).

The scanned skull samples were then decalcified and paraffin embedded. Tissue sections measuring 5 µm thick were cut from the paraffin blocks and stained with hematoxylin and eosin (H&E) and Masson’s trichrome. Micrographs of the stained sections were taken using an optical microscope (Olympus BX51, Japan) equipped with a digital camera (Olympus DP72).

Statistical Analysis

All measured values were compiled into respective data array and analyzed using IBM SPSS Statistics 25 software. One-way analysis of variance with post hoc multiple comparisons was used to determine significant differences between groups.

Results

Figure 1 shows the SEM micrographs chitosan hydrogel cross-section (Figure 1A), surface of Gelatin-BCP electrospun mat with EDS profile (Figure 1B), cross-section of the chitosan/gelatin-BCP composite (Figure 1C) and high magnification cross sections of the chitosan layer (Figure 1D) and gelatin-BCP (Figure 1E). Chitosan hydrogel pore size measure approximately 126 ± 35.72 µm in diameter while fiber diameter of the gelatin-BCP mat had an average size of 14.25 ± 7.92 µm. Electrospun fiber morphology is quite irregular due to incorporation of the BCP powder within the fibers. Presence of BCP powder was confirmed through the examination the fibers EDS profile showing presence of calcium. The thickness of the electrospun layer was measured to be at an average of 331.16 ± 13.016 µm.

Figure 1.
Figure 1.:
Chitosan hydrogel cross-section (A), surface of Gelatin-BCP electrospun mat (B) with EDS profile, cross-section of the chitosan/gelatin-BCP composite (C) and high magnification cross sections of the chitosan layer (D) and gelatin layer (E). BCP, biphasic calcium phosphate; EDS, energy dispersive spectrometry.

To assess the cytocompatibility of the bi-layer composite material, proliferation and attachment behavior of MC3T3-E1 cells were analyzed though MTT assay and confocal microscopy. Figure 2A shows the MTT data retrieved from M3CT3-E1 cell cultured on samples of chitosan/gelatin-BCP composites. Both the electrospun gelatin and the composite material were able to support preosteoblast cells within the 1 week period with significant increase in cell number across all observation periods with no significant difference in cell number after 1–3 days of cell culture (P < 0.05). Results show that at 5 days, cell number was significantly higher on the chitosan/gelatin-BCP (1.63 ± 0.06) compared with that of the gelatin (1.41 ± 0.08; P < 0.05). This was also true at 7 days of culture in which the gelatin (1.73 ± 0.13) had significantly lower cell number in comparison to chitosan/gelatin-BCP (2.16 ± 0.06; P < 0.01). This is further confirmed through the examination of the confocal micrographs. Seeded cells after 1 day (Figure 2B), 3 days (Figure 2C), 5 days (Figure 2D), and 7 days (Figure 2E) were visualized by fluorescent staining. Extensive attachment and increased size is very noticeable between 3 days and 7 days after seeding.

Figure 2.
Figure 2.:
Cell proliferation data based on MTT assay of MC3T3-E1 cells seeded on composite chitosan/gelatin-BCP hemostat and gelatin electrospun mat (A). Number of cells seeded on composite chitosan/gelatin-BCP was significantly higher after 5 and 7 days (*P < 0.05; **P < 0.01; n = 3 per group). Confocal micrographs of MC3T3-E1 cells showing attachment behavior and proliferation after 1 day (B), 3 days (C), 5 days (D), and 7 days (E). BCP, biphasic calcium phosphate.

Effective blood absorption is an important factor to consider in developing a topical hemostat. To test the absorption capability of the chitosan/gelatin-BCP composite material, an ex vivo blood absorption test was done using acid citrate anticoagulated blood samples extracted from Sprague-Dawley rats. Figure 3A shows the absorption data and a section of a sample used for blood absorption test stained with H&E. Statistical analysis of the measured absorption values measured at 540 nm indicates significant increase in absorbed amount of blood over the course of 1- to 5-minute test (P < 0.05). The data indicates that the chitosan/gelatin-BCP composite is capable of absorbing blood beyond three minutes. Stained sections of the samples used in the blood absorption test show the absorption behavior of the bi-layer composite material. The cross-section (Figure 3B) of the sample shows that main cellular components of the blood are highly concentrated within the electrospun gelatin-BCP layer which is the main contact point for the blood. The hydrogel layer composed of chitosan (Figure 3C) barely contains cellular components aside from its fraction nearest to the electrospun layer. High magnification examination of the electrospun layer (Figure 3D) reveals the aggregation of red blood cells between the gelatin-BCP fibers.

Figure 3.
Figure 3.:
Blood absorption data of composite chitosan/gelatin-BCP hemostat using anticoagulated rat blood (A) showing significant increase in absorbed blood after 1, 3, and 5 minutes (*P < 0.05; n = 12 per group). Cross-section of composite chitosan/gelatin-BCP hemostat sample (B) used in the blood absorption test. High magnification of the chitosan hydrogel layer (C) and gelatin-BCP electrospun mat (D). BCP, biphasic calcium phosphate.

Usability of the chitosan/gelatin-BCP composite material as a hemostat for bleeding bone defects was also investigated through topical administration of samples and degradable hemostat testing in noncritical sized defects. Figure 4 shows the samples for degradable hemostat testing (Figure 4A) and for topical hemostatic application (Figure 4B). Morphology of the surgical sites before suturing can be seen in Figure 4 C1–C4 Figure 5A shows the three-dimensional Micro CT reconstructions of the rat skull defect 1 and 3 weeks after surgery. Based on the reconstructed images and calculated data, using the chitosan/gelatin-BCP composite material improved bone regeneration. Figure 5B shows the three-dimensional bone volume analysis of the reconstructed data sets of 1 week and 3 weeks skull samples. Statistical analysis of the quantified bone volume over tissue volume indicates that the control (7.98 ± 0.72) was significantly lower compared to BWS (12.75 ± 1.94), topical hemostatic test (14.58 ± 3.13) and the degradable hemostat group (15.67 ± 3.98) (P < 0.05) after 1 week. At three weeks, bone regenerated in animals in which BWS (13.98 ± 2.40) was used did not significantly differ compared to the control (18.34 ± 9.42) (P < 0.05). Both hemostat (26.44 ± 4.82) and degradable hemostat (21.79 ± 4.24) groups had significantly higher volume of regenerated bone (P < 0.01) compared with the control and BWS group. Bone growth in defects with either topical hemostat application or degradable hemostat had significantly consistent increase in bone regeneration compared to the control defect.

Figure 4.
Figure 4.:
Images of 3 mm diameter samples for implant procedure (A) and 10 mm diameter samples for hemostat testing (B). Hemostat testing (C) was done by drilling a 3 mm defect (C1) until bleeding was achieved (C2) then sample was applied (C3) until bleeding is stopped. A control defect is also created on the other side of the skull (C4).
Figure 5.
Figure 5.:
Three-dimensional reconstruction of analyzed micro-computed tomography (microCT) data (A) and Bone volume/Tissue volume analysis (B) of extracted rat skulls tested with composite chitosan/gelatin-BCP (topical hemostat), composite chitosan/gelatin-BCP (degradable hemostat), bone wax substitute (BWS), and as bleeding control. Results indicate improved bone regeneration after 1 week when using hemostatic materials (*P < 0.05) while significantly higher bone formation after 3 weeks when using composite chitosan/gelatin-BCP as either a topical hemostat or degradable hemostat (**P < 0.01). Averaged measurements were taken from three animals per group, per specified observation period. BCP, biphasic calcium phosphate.

Effective bone regeneration due to hemostatic application or implantation of the chitosan/gelatin-BCP was further confirmed through the inspection of tissue sections from the skull samples. Figure 6 shows the H&E stained tissue sections of a control defect (Figure 6 A-A2 and D-D2), hemostat test (Figure 6 B-B2 and E-E2) and implant (Figure 6 C-C2 and F-F2) after 1 week and 3 weeks. One week after hemostatic application and implantation of the chitosan/gelatin-BCP samples, there is minimal difference between the control and the treated defects. Majority of the growth within the defects are fibrous tissue (Figure 6 A1, B1, C1). Bone formation has already started along the borders of the bone defect in all samples (Figure 6 A2, B2, and C2). Implanted samples of the bi-layer composite material resulted in more extensive fibrous tissue growth and vascularization intertwined with the remaining material (Figure 6 C1). Three weeks after the surgical procedures and testing, the control setup (Figure 6 D2) shows advanced regeneration of bone tissue from the edges of the defect with a thinner fibrous tissue bridging the gap between the two points (Figure 6 D1). Both hemostatic application (Figure 6E) and implantation (Figure 6F) of chitosan/gelatin-BCP have noticeably enhanced bone regeneration in the treated defects. Extensive cartilage-bone tissue can be found within the defect area (Figure 6 E1 and F1) and the defect edge (Figure 6 E2 and F2).

Figure 6.
Figure 6.:
Tissue sections stained with hematoxylin and eosin showing bone tissue regeneration of control defect (A1 to D2), hemostat tested defects (B and E2) and implanted defects (C and F2) 1 week and 3 weeks after surgery. (Inset box shows regions of high magnification images (A1–F2).

Discussion

Hemorrhage could ultimately affect tissue regeneration, lead to serious complications and even death. This condition is naturally controlled through an intrinsic mechanism called hemostasis which prevents too much blood loss. This can be attained through several factors that activate the coagulation cascade of the blood to form a stable platelet plug or through the occlusion of vessels once blood flow is diminished or pressure is applied.1–3 Bone bleeding is particularly difficult to stop due to the inherent stability of the tissue structure which does not allow the collapse of the source of bleeding thus maintaining the flow and in turn preventing the formation of a stable plug. Thus, the most effective way to impede blood loss in bone bleeding is through application of absorbent materials such as cotton or bone wax; however, application of these materials could affect bone healing.9–11

In this study, a bi-layer composite hemostatic material was designed, fabricated and tested specifically for bone tissue application. The fabrication involved two of the most common methods for fabricating scaffolds for tissue engineering; freeze-dried hydrogels and electrospinning. Chitosan hydrogel was fabricated through dissolution with 1% acetic acid, freezing then freeze drying to form a porous structure after which a layer of gelatin-BCP was deposited on one side of the hydrogel via electrospinning. Because both chitosan and gelatin are highly biodegradable14,17,18 there is a need to stabilize these components for the bi-layer composite material to be applied as a hemostat. Cross-linking both components using EDC-NHS has already been noted in several researches to be highly effective and has minimal effect on biocompatibility of the resulting material, making it a suitable crosslinking agent for the bi-layer composite hemostat material.25–27 Biphasic calcium phosphate was incorporated into the gelatin electrospun layer to impart osteoconductivity and protein adsorption.28–31 As mentioned, hemostasis in bone is mainly realized by application of highly absorbent material or placement of a suitable material barrier what can counter act the out flow of blood from its source.

The composite chitosan/gelatin-BCP material was designed to absorb blood and create the said artificial barrier as seen in the examination of its hemostatic activity and tissue sections of samples retrieved from blood absorption tests. Figure 7 shows the mechanism by which chitosan/gelatin-BCP initiates the hemostasis. The gelatin-BCP layer, being the section of the material directly in contact with blood, acts as a filter which accumulates the blood cellular components. The fibrous mesh typically swells upon contact with the fluid component of blood, reducing its permeability to red blood cells. This also helps in the transition of the blood serum into the chitosan hydrogel layer. Due to its three-dimensional porosity, the chitosan hydrogel layer acts as a sponge, capable of greater fluid uptake compared to the fibrous gelatin layer.32–34 This absorptive property assists in the build-up of cellular components on the electrospun layer and enables the stabilization of the thin fibrous layer which would otherwise disintegrate upon saturation with fluids (Figure 3B and Figure 7). By filtering and aggregating majority of the cell components along the electrospun layer of the composite chitosan/gelatin-BCP material would then aid in the formation of a stable plug to prevent further bleeding on the bone defect. Although the composite material is a solid mass and would not perfectly fit into the bleeding bone channels, its mechanism of building up blood cell material eventually creates a stable clot mass which occupies the minute gaps between the bone and the material and eventually occludes the bleed site (see Supplemental Digital Content, http://links.lww.com/ASAIO/A307).

Figure 7.
Figure 7.:
Schematic diagram showing the hemostatic mechanism of the composite chitosan/gelatin-BCP upon contact with blood. Whole blood containing serum and cells comes in contact with the fibrous electrospun layer (1). Both the electrospun layer and the hydrogel absorbs most of the serum and fluid blood components (2). Both layers continues to absorb the fluid blood components and the electrospun fibers begin to swell trapping the cells (3). Swollen fibers filter out most of the cellular components while serum is continuously absorbed within the hydrogel layer (4). BCP, biphasic calcium phosphate.

Frontal skull plates were removed from the treated rats 1 and 3 weeks after sugery. Micro CT analysis of the skull plates indicates that using chitosan/gelatin-BCP composite material either as a topical removable bone hemostat or a degradable hemostat resulted to improved bone regeneration in noncritical sized defects. Application of the composite chitosan/gelatin-BCP as a biodegradable hemostatic material consequently improved bone regeneration when compared to the control or a commercially available BWS (Ostene). This was exemplified through the examinations of tissue sections and calculation of the bone volume fraction after 3 weeks of application of the material as either topical hemostat or as a degradable hemostat.

Conclusion

A bi-layer composite topical hemostat composed of chitosan hydrogel and gelatin-BCP electrospun mat was successfully fabricated and tested on bleeding bone. Successful hemostasis was achieved using the bi-layer composite material through the filtration of cells on the electrospun layer and continuous absorption of blood serum within the hydrogel layer. The bi-layer composite hemostat exhibited good in vitro biocompatibility and improved bone regeneration on noncritical rat calvarial defects.

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Keywords:

bone; topical hemostat; chitosan; gelatin; biphasic calcium phosphate

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