Research ArticleExperimental Studies

Performance of Novel Nanofibrous Biopolymer Membrane for Guided Bone Regeneration within Rat Mandibular Defect

JOONG-HYUN KIM, MI-KYUNG KIM, JEONG-HUI PARK, JONG-EUN WON, TAE-HYUN KIM and HAE-WON KIM
In Vivo July 2011, 25 (4) 589-595;

Abstract

Background/Aim: A novel nanofibrous membrane of a degradable biopolymer poly (lactide-co-ε-caprolactone) (PLCL) for guided bone regeneration (GBR) was designed and its tissue compatibility and ability to promote the regeneration of new bone were investigated in a rat mandibular defect model. Materials and Methods: The nanofibrous structuring of the PLCL polymer was facilitated by a solvent-induced phase separation method using camphene as the porogen. The PLCL membrane was implanted in a critical-sized (5 mm diameter) defect of the rat mandible. Results: The assessment of cell compatibility conducted using undifferentiated pre-osteoblast cells (MC3T3-E1) showed favorable cell adhesion and growth on the nanofiber PLCL membrane with an active cytoskeletal processes and increment in the cell population with culture time. In vivo results at four weeks post-operation demonstrated that the PLCL nanofibrous membrane induced better guided new bone formation than the defect control group while protecting the bone defect against the ingrowth of fibrous tissues. Conclusion: Based on these results, the newly-developed PLCL nanofibrous biopolymer may be useful as a biocompatible and bone regenerative guidance membrane in dentistry.

The rapid appearance of fibrous connective tissues in bone defects has generally been considered a major problem in the healing process of the periodontal pocket (1, 2). The guided bone regeneration (GBR) approach has commonly been applied to generate an optimal environment to secure the physical space and improve the intrinsic growth capacity of bone ingrowth into the defect area (3, 4). The application of a suitable device, generally in the form of a thin membrane, facilitates the formation of neo-bone tissues while preventing the ingrowths of other tissues, which would hamper the expected bone regeneration. Many studies have already confirmed that the use of a membrane provided an effective mechanical barrier against the formation of fibrous connective tissues while permitting osteogenesis to occur (2, 3, 5-9). Various GBR membranes have been investigated for the treatment of periodontal pocket and long bone defect with nonunion (1, 10, 11).

Different types of biomaterials have recently been developed to retain the physicochemical and mechanical properties that are suitable for use as a GBR membrane (10, 12-16). There are many factors to consider in the design and synthesis of GBR membranes, primarily including biocompatibility and physico-mechanical stability (17). Among the currently available designs, those with a porous structure have largely shown better bone formation within the defect area when compared to those with a dense structure (17). Bioabsorbable materials are favored because they do not require second surgery for removal of the membrane after bone regeneration. Accordingly, an appropriate degradation rate is considered an important index for the clinical application of such materials. Moreover, GBR membranes with selective permeability are favored because they prevent unexpected tissue invasion while allowing nutrient and oxygen supplies (10, 18).

In the present study, the suitablilty of a degradable polymer membrane retaining a nanofibrous structure for the purpose of securing a space for bony tissue ingrowth with nutrients and oxygen supply while preventing the invasion of fibrous connective tissues was investigated. As a degradable biopolymer, poly (lactide-co-ε-caprolactone) (PLCL), a copolymer of PLA (poly(L-lactide)) and PCL (poly(ε-caprolactone)), was used due to its biocompatibility and appropriate biodegradability as well as elastic properties and moldability (19-21). By varying the composition of monomers and modifying the fabrication procedures, the mechanical and physical properties can also be tuned to the required properties for GBR membranes. Specifically, the degradable polymer was produced in the form of a nanofibrous membrane that was generated using a novel solvent-induced phase separation technique employing camphene (21-23). The in vivo tissue compatibility and performance in guiding bone regeneration of the nanofibrous PLCL membrane was then investigated within a critical-sized defect of the rat mandible.

Materials and Methods

Preparation of nanofibrous biopolymer GBR membrane. The PLCL (poly (L-lactide-co-ε-caprolactone); Boehringer Ingelheim, Ingelheim, Germany), which is a co-polymer of 70% PLA and 30% PCL was used to produce the membrane. While PLA is a crystallizable material that is hard and brittle PCL is a semicrystalline material with rubbery properties; therefore, the co-polymers exhibit variable mechanical properties depending on their composition. The PLCL was dissolved in chloroform and mixed with camphene (C6H16, Sigma-Aldrich, St Lois, MO, USA), which was used as a porogen. Samples without camphene were also prepared for comparision. The ratio of PLCL-to-camphene was set at 1:14 to produce a nanofibrous structure. The mixture was poured into a Teflon mold, allowed to solidify under ambient condition and fully dried in a freeze dryer. The two phases of PLCL and camphene form a bicontinuous network by the solvent-induced phase separation process and, due to the ease-of-sublimation of camphene under ambient conditions, the remnant PLCL network becomes highly porous and even nanofibrous when there is appropriate camphene content (23). The morphology of the membranes was investigated by scanning electron microscopy (SEM) after gold coating the samples.

Osteoblast proliferation assay on the GBR membrane. In vitro tests on the nanofibrous PLCL membrane were conducted using an undifferentiated pre-osteoblast cell line (MC3T3-E1). The GBR membranes were sterilized by ethanol under UV light and then rinsed three times with phosphate buffered saline (PBS). The osteoblast culture was maintained in complete medium that contained α-minimal essential medium (α-MEM), 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C under 5% CO2. The osteoblast cells were then detached using 0.2% trypsin EDTA and the number of cells was counted using a hemocytometer. The cells were seeded on the nanofibrous PLCL membrane at a density of 3×104 and the samples were then incubated in complete medium. The proliferation of attached cells was measured by MTS using an assay kit (CellTiter 96 Aqueous One Solution, Promega, Madison, WI, USA) (24). After culture for 1, 3 or 7 days, the samples were washed three times with PBS. Next, MTS reagent was added to each sample which was then incubated for 2 h at 37°C. Aliquots of the solutions were subsequently transferred to 96 well plates and the absorbance was measured at 490 nm using a microplate reader (iMark, BioRad, Tokyo, Japan). The cell attachment and growth of the samples were observed by SEM. Cells grown on the sample were washed twice with PBS and subsequently fixed with 2.5% glutaraldehyde. Next, the samples were rinsed with distilled water and dehydrated with graded concentrations of ethanol. Finally, the samples were treated with hexamethyldisilazane and coated with gold.

Animal surgery for GBR within a rat mandible. A total of 10 male Sprague-Dawley (SD) rats (10 weeks old, body weight approximately 250 g) were selected to evaluate the GBR behavior of the nanofibrous PLCL membrane. The animals were housed in plastic cages under a 12 h-day/night cycle and an ambient temperature of 21°C. The animals were allowed access to water and standard laboratory pellets ad libitum. The protocols used in this study were approved by the Dankook University Institutional Animal Care and Use Committee, Korea.

Prior to surgery, the animals were anesthetized with an intramuscular injection of ketamine and xylazine complex. Local anesthetic (0.5 mL 2% lidocaine) was administered at the operation site prior to incision. The lower jaw areas were shaved and prepared with 10% povidone iodine and 70% ethanol. After disinfection of the skin, a linear incision was made at the midline. Following incision of the muscle along the submandibular border, mucoperiosteal flaps were raised bilaterally at both the buccal and the lingual sides of the bone around the mandibular angle. Care was taken not to injure the facial nerve and parotid duct. Next, a 5 mm diameter trephine burr was used to create a standard full-thickness round defect on both right and left sides of the mandibular angle. During generation of the defect on the mandible, the surgical field was continuously irrigated with cooled physiological saline to reduce thermal damage. Subsequently, the defect was covered with the membrane (12 mm in diameter), and the animals were then allowed to heal for four weeks. Defects without the membrane were also examined as a control group. When membranes were used, they were trimmed to extend about 2 mm outside the margins of the defect, adapted to the surrounding bone and then fixed to nearby soft tissue using absorbable sutures. The mucoperiosteal flaps were carefully repositioned on the outer side of the mandible and sutured with absorbable suture. Finally, the skin was sutured for total coverage with non-absorbable sutures. The rats were housed individually.

Histology and micro-computed tomographic evaluation. At four weeks after surgery the animals were sacrificed. Each mandible was explanted with the surrounding soft tissues. All the specimens were radiographically examined using a digital dental X-ray unit (ADX4000, Dexcowin, Seoul, Korea) with an exposure time of 0.1 sec (60 kVs, 1 mA). The explanted parts of the mandible were then placed on a 3×4 cm digital CCD (charge coupled device) sensor. For histology and micro computed tomographic (μCT) analysis, the specimens were fixed by immersion in 10% neutral buffered formalin solution. After 48 h fixation, the samples were rinsed with saline and imaged by μCT (Skyscan-1172, Skyscan, Kontich, Belgium). The samples were then dehydrated in an ethanol series and decalcified in RapidCal sol (BBC Biochemical, Stanwood, WA, USA). Excess muscle was then removed from the specimens using a scalpel and the specimen was then sawed in half. Sections with a thickness of 5 μm were then cut from one half of the embedded specimen using a microtome. Finally, the sections were stained according to the traditional hematoxylin and eosin and Masson's trichrome methods.

For analysis of the μCT data, the amount of defect healing was expressed as the percentage of defect closure using image analysis software. First, based on the difference in grey values, the individual threshold of the bone/no-bone boundary was determined for each digitized microradiograph. This threshold was then applied to the entire 5.0 mm diameter defect and the remaining defect area was measured automatically. Finally, this remaining defect area was expressed as a percentage of the original defect size. After the measurements were completed, the percentage of the average defect closure was calculated for the three experimental groups. Image analysis software (Scion image, NIH) was used to measure the newly formed bone area histomorphometrically.

Figure 1.
Figure 1.

SEM morphology of the PLCL nanostructures produced without (a) or with (b) the porogen camphene (1:14, PLCL to porogen).

Statistical analysis. The mean and standard deviation values for each parameter were calculated for each of the different groups. Differences in the average percentage of bone formation between the groups were determined by Student's t-test at a significance level of p<0.05.

Results

Morphology of the nanofibrous PLCL membrane and in vitro cell growth. Figure 1 shows the SEM images of the PLCL membranes produced with or without the use of porogen camphene. The PLCL membrane fabricated without camphene had a compact, dense and smooth surface (Figure 1a). However, when camphene was added to the PLCL composition during the preparation of a membrane, a highly porous nanofibrous structure was produced (Figure 1b).

Figure 2.
Figure 2.

Proliferation of MT3T3-E1 cells on the nanofibrous PLCL membrane (a), as measured by MTS assay, and SEM cell growth morphology on the nanofibrous PLCL membrane surface after culture for 7 (b) and 14 (c) days.

The in vitro cell viability on the PLCL membrane was briefly assessed, as shown in Figure 2. By the MTS method, the cell growth increased during culturing up to 7 days, showing good cell proliferation on the nanofibrous membrane (Figure 2a). As observed by SEM at days 7 and 14 (Figures 2b and 2c), a large number of cells had spread on the nanofibrous membrane with highly elongated cytoskeletal extensions. Cell confluence was observed to occur at around day 14, and some cells were even shown to migrate into the pore channels of the nanofibrous network.

In vivo tissue responses to the nanofibrous PLCL membrane. There were no side-effects and wound healing was uneventful in all the rats and no noticeable host rejection responses were observed. The sutures were removed on the tenth day after surgery and no marked inflammatory reactions were observed in the animals that underwent surgery throughout the study period. The masticating ability of each animal gradually recovered to normal over two days.

In all the defect sites, the nanofibrous membranes were well placed, adapted and tolerated by the animals. Representative radiograph and μCT images of the defect location from a PLCL membrane bearing animal are shown in Figure 3. μCT images of the new bone formation in the PLCL membrane and blank control groups are shown in Figure 4. New bone formation toward the center of the defect between two membranes was clearly observed in the PLCL group (Figure 4b). In contrast, the control group showed new bone formation with an irregular direction from the border of the defect, not toward the center of the defect (Figure 4a). Based on the μCT analysis the newly-formed bone volume on the nanofibrous PLCL membrane group was 11.53±1.83%, which was not significantly different from the control group (12.05±1.29%). However, the bone surface of the PLCL membrane group (11.26±1.41) was significantly higher (p-value=0.022) than that of the control group (9.24±0.47).

Figure 3.
Figure 3.

Radiographic (a) and μCT (b) images of a rat mandible where PLCL membrane was used to cover the defect. The original outline of the 5 mm defect is clearly visible, and two horizontal lines indicate the defect margin. Micro-CT image shows new bone formation within the defect area at four weeks post-operation.

Figure 4.
Figure 4.

Representative μCT images of new bone formation within the mandible defect area: blank control group (a) and PLCL membrane group (b), taken at 4 weeks post-operation. Arrow: direction of new bone growth; White line: defect margin.

Histological slides from the nanofibrous PLCL membrane group showed prominent new bone formation along the margin of the pre-existing bone. New bone was shown to form along the membrane from the margin to the center of the defect and no connective tissue was observed in the defect area (Figure 5a). New bone formation was also observed in the control group, but the growth was not toward the defect, but rather in an irregular direction (Figure 5b). Connective and muscular tissue was observed within the defect area. The percentage of new bone area in the nanofibrous PLCL membrane group amounted to 56.84±3.84% of the total defect area after four weeks of healing. Newly formed bone tissue was distributed over the entire surface of the defect margin. The new bone area of the control group corresponded to 44.01±6.56%, which was significantly different (p-value=0.028) from the membrane group. The length of new bone growing in the membrane group (58.58±6.71%) was also significantly higher (p-value=0.049) than that in the control group (42.47±10.37%).

Figure 5.
Figure 5.

Representative histological appearance of the rat mandibular defect at 4 weeks post-operation. PLCL group showing new bone formation following the membrane (a). Control group also showing new bone formation, but lacking directionality (b). Arrows: defect margin; Dashed arrows: direction of new bone growth; OB: Old bone; NB: Newly formed bone. H & E stain, ×40.

Discussion

Here, we developed a novel type of biopolymer membrane with a nanofibrous structure that was specifically produced by the solvent-induced phase separation method using a novel porogen, camphene. Because the camphene and PLCL solution easily solidified upon chloroform evaporation, characteristic structure known as a bi-continuous phase was formed between PLCL and camphene. Considering that the electrospinning process is commonly used to obtain a nanofibrous polymer membrane, this novel nanofiber-structuring technique is very useful due to its ease and simplicity, finding an alternative to the electrospinning process (23).

This newly-developed nanofibrous PLCL membrane is considered to have the capacity to create and maintain adequate space for GBR. Moreover, its nanofibrous structure may provide a channel for nutrients and oxygen supply because of the porous structure. The number of viable cells increased over the seven days of culture, during which time the cell morphology also showed good adhesion, spreading and population of cells, suggesting that the PLCL membrane possessed in vitro biocompatibility and may be useful as a cell supporting and guiding matrix in tissue regeneration. Many recent studies have been conducted to evaluate cell responses to nano-structured and/or nanofibrous-structured surfaces (25). For example, Fujihara et al. reported that cell attachment and proliferation were enhanced on a nano-structured surface when compared to a micron-structured surface (3). Others have found that polymer nanofibers made by electrospinning were attractive for the growth of various types of cells including fibroblasts, mesenchymal stem cells, nerve cells, chondrocytes, smooth muscle cells and endothelial cells (26-32).

The 5 mm diameter of the defect created in the present mandibular defect model is considered to be a critical size for evaluation of the bone healing effect of the GBR membrane (33). When the nanofibrous PLCL membrane was used, healing was uneventful in all the animals with no evidence of inflammation in the surrounding tissues. The μCT allowed clear visualization of new bone formation within surrounding tissues and old bone (2), and thin radiolucent lines between the old and newly formed bone indicated that new bone formed in the defect area. A substantial reduction in defect size was indicated in both the nanofibrous PLCL membrane and blank control groups, and the new bone growth occurred in a specific direction, i.e. toward the center of the defect in the membrane group, in contrast, to the irregular direction of new bone formation in the control group. The nanofibrous PLCL membrane was thus considered to provide an optimal environment for the intrinsic growth ability of bone tissue into the defect area.

The main biological function of GBR membranes is to provide a physical barrier against the surrounding environment, which both prevents the ingrowth of soft tissue and the loss of intrinsic healing factors, consequently promoting bone tissue ingrowth (10). The nanofibrous PLCL membrane used here was tightly fixed by suturing to prevent the ingrowth of soft tissues into the gap between the barrier and the bone surface (3, 34, 35). Histologically, no invasion of fibrous connective tissues into the defects was demonstrated when they were protected by the nanofibrous PLCL membrane, but conversely, in the completely open control defects, noticeable level of ingrowth of soft tissues, muscle and connective tissues into the defect area in combination with newly formed bone was shown.

Although the fibrous connective tissue that grew into the defect area did not greatly disturb the amount of new bone formation, the control group showed large variations in the volume of the bone. Lundgren et al. (17) also revealed a conspicuous pattern of uneven distribution of bone tissue and ingrowth of suprabony connective tissue. Kellomäki et al. (36) using a porous membrane inserted in a bone defect reported directional bone growth mainly by osteoblasts in close contact with adjacent bone, suggesting that the osteoblasts can be switched to bone tissue when the membrane is degraded. In the present study, the nanofibrous membrane was also found to be filled with cells, extracellular matrices and mineralized bone. Since nanofibrous substrate can effectively direct and populate tissue cells and trigger their secretion of an appropriate tissue matrix, leading to recovery of the function of damaged tissues (37), the osteoblasts accumulating in the defect region around the nanofibrous membrane should also play a role in promoting mature bone with a prolonged implantation time.

One possible limitation of the nanofibrous PLCL membrane in the GBR applications was its mechanical stability. While placement in the appropriate defect positions and suture processes were possible, the nanofibrous membrane was not strong enough to bear mechanical loads such as titanium mesh or polytetrafluoroethanol (PTFE). Therefore, nanofibrous PLCL membrane applications may be limited to the treatment of defects in which mechanical loading is not an issue. Accordingly, strengthening of the nanofibrous PLCL membrane may be a challenge to address in future studies.

Conclusion

The newly-developed nanofibrous PLCL membrane for guided bone regeneration within a rat mandibular defect provides a tissue compatible substrate with no rejection response. The nanofibrous PLCL membrane is considered to have the potential for use as a GBR membrane.

Acknowledgements

This work was supported by the Priority Research Centers Program (grant#: 2009-0093829) and WCU (World Class University) program (grant#: R31-10069) through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology.

  • Received January 6, 2011.
  • Revision received March 13, 2011.
  • Accepted March 14, 2011.

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Performance of Novel Nanofibrous Biopolymer Membrane for Guided Bone Regeneration within Rat Mandibular Defect
JOONG-HYUN KIM, MI-KYUNG KIM, JEONG-HUI PARK, JONG-EUN WON, TAE-HYUN KIM, HAE-WON KIM
In Vivo Jul 2011, 25 (4) 589-595;
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