Chitosan nanofiber membranes have been known to have a high degree of biocompatibility and support new bone formation with controllable biodegradation. The surface area of these membranes may allow them to serve as local delivery carriers for different biologic mediators. Simvastatin, a drug commonly used for lowering cholesterol, has demonstrated promising bone regenerative capability. The aim of this study was to evaluate simvastatin loaded chitosan nanofiber membranes for guided bone regeneration (GBR) applications and their ability to enhance bone formation in rat calvarial defects. Nanofibrous chitosan membranes with random fiber orientation were fabricated by electrospinning technique and loaded with 0.25 mg of simvastatin under sterile conditions. One membrane was implanted subperiosteally to cover an 8 mm diameter critical size calvarial defect. Two groups : 1) Control: non-loaded chitosan membranes ; 2 ) Experimental: chitosan membranes loaded with 0.25 mg of simvastatin were evaluated histologically and via micro-computed tomography (micro-CT) for bone formation at 4 and 8 week s time points (n = 5/group per time point). Both groups exhibited good biocompatibility with only mild or moderate inflammatory response during the healing process. Histologic and micro-CT evaluations confirmed bone formation in calvarial defects as early as 4 weeks using control and experimental membranes. In addition, newly-formed bony bridges consolidating calvarial defects histologically along with partial radiographic defect coverage were observed at 8 weeks in both groups. Although control and experimental groups demonstrated no significant statistical differences in results of bone formation, biodegradable chitosan nanofiber membranes loaded with simvastatin showed a promising regenerative potential as a barrier material for guided bone regeneration applications.
Guided bone regeneration (GBR) technique is a widely utilized surgical approach in the augmentation of alveolar bone deformities that are frequently observed in edentulous patients. A wide range of nonresorbable and resorbable barrier membranes are currently used in GBR procedures to prevent soft tissue infiltration and achieve osseous tissue formation [
Chitosan is a natural co-polymer composed of N-acetyl-glucosamine and glucosamine units, and has been investigated as an alternative material in bone tissue regeneration due to its well described biocompatible and biodegradable properties [
Many studies examining chitosan membranes have focused on the incorporation of growth factors such as bone morphogenetic proteins (BMPs) and platelet derived growth factor (PDGF) to enhance bone regeneration in GBR applications [
Simvastatin, an affordable and widely used hypolipidemic agent in the management of cardiovascular diseases, may be a promising alternative to these growth factors for bone healing stimulation. Early human studies reported an association between systemic statin use and reduced risk of hip fracture as well as increased bone density [
In recent years much attention has been focused on local delivery strategies of simvastatin to allow adequate dosage at the desired site and avoid systemic side effects such as liver toxicity and myositis. Locally delivered simvastatin is reported to positively influence bone regeneration in the treatment of periodontal disease, maxillary sinus augmentation and enhance osseointegration around dental implants [
By taking advantage of drug loading and local delivery potential of nanofiber chitosan membranes with the potential of simvastatin as an alternative to expensive growth factors, the ability of chitosan nanofiber GBR membranes to support bone regeneration in alveolar bone deformities may be enhanced. Therefore, the aim of this study was to evaluate simvastatin loaded chitosan nanofiber membranes ability to enhance bone formation in a critical sized rat calvarial defect model.
Nanofibrous chitosan membranes with random fiber orientation were fabricated by electrospinning a 5.50 wt% chitosan (degree of deacetylation (DDA) = 70% deacetylated, molecular weight (MW) = 311.5 KDa, Primex) in 70% trifluoroacetic acid and 30% methylene chloride solution in a syringe with a blunt 20 gauge needle, at 25 kV voltage as previously described [
The addition of the butyric fatty acid molecule to the outside of the chitosan nanofibers increases the hydrophobic characteristics of the membrane and retains the nanofiber morphology when immersed in aqueous solutions [
In this study, two nanofiber chitosan membranes were compared: 1) plain chitosan membranes as control; and, 2) chitosan membranes loaded with 0.25 mg of simvastatin/implant as experimental group. All animal procedures were in compliance with the Guide for the Care and Use of Laboratory Animals and the
University of Memphis Institutional Animal Care and Use Committee (Protocol No. 0747). A critical size calvarial defect of 8 mm in diameter was created in 25 Sprague-Dawley rats (approximately 2 months of age and 370 g). Animals were randomized into 4 groups (n = 5/group per time point). 1 - 2 animals were added to each group to compensate for sample attrition due to death of animals. These animals were selected because they provide adequate size and tissue volume for testing the membranes, and are widely accepted for determining wound healing and tissue response to the implanted membranes. Briefly, rats were anesthetized with an inhalation of isoflurane (1%) in an oxygen carrier. A midline incision in the skin over the cranium was made from the middle of the nasal bones to the posterior nuchal line (~20 mm). The underlying soft tissue and periosteum were incised and reflected to expose the calvaria. An 8-mm diameter circular craniotomy was made in the center of the exposed calvaria using a custom trephine bur (
Retrieved calvarial samples were fixed in 10% neutral buffered formalin for 72 hours and transferred to phosphate-buffered saline (PBS). High resolution micro-CT was utilized to scan and evaluate the healing and mineralization of the
calvarial defects. The entire heads were scanned in a 12.3-mm diameter sample holder at 8 μm resolution, energy level of 55 kV, and intensity of 72 μA on a μCT40 scanner (Scanco Medical, Basserdorf, Switzerland). Data were reconstructed using the Scanco Imaging processing software for further morphometric and density analysis. Reorientation of the reconstructed micro-CT graphs was done using data viewer software (Bruker AXS Inc.) Volume of Interest was identified as a cylinder that corresponded and overlapped the original defect’s shape, volume and location. Height of cylinder was extended 0.1 - 0.2 mm superior and inferior to defect to allow for accurate measurements [
Previously scanned samples were then decalcified and prepared for paraffin embedding and staining with hematoxylin and eosin (H&E). Embedded samples were sectioned in the sagittal direction into 4 μm serial slices through the middle of the calvarial defect. Hematoxylin and eosin staining provided a general histological overview of membrane shape and structure, new bone formation in defect and appearance of surrounding soft tissues. Sections were viewed and inflammatory reaction was graded by a blinded pathologist using a 4-point scoring system (0 = absent, 1 = mild, 2 = moderate, and 3 = severe) to determine the tissue reaction to the membranes and to observe membrane degradation [
Ninety-five percent confidence intervals were calculated and used to compare the ordinal inflammatory response scores. Two-way ANOVA at the 0.05 level of significance was used to compare the mean values of the histologic and micro CT measurements in the control and experimental groups at 4 and 8 week time points.
Surgical procedures were performed without complications on 21 rats and animals recovered well after surgery. Four rats were lost following surgery due to post-operative bleeding and intra-operative brain tissue trauma. For surviving animals, calvarial wounds healed predominantly uneventfully without showing clinical signs of inflammation. Primary closure of wound area was maintained throughout the experiment. Following retrieval, visual examination of the specimens revealed membranes that were still present covering the defect in the rat calvaria (
In general, no adverse histological reaction was observed in the tissues surrounding the implanted membranes. At 4 weeks most specimens in both groups showed mild granulation tissue response with mainly neutrophilic migration towards the membrane surface along with few foreign-body giant cells (
The inflammatory response against these materials was assessed using the 4-point scoring system. As shown in
Membrane Type | Confidence Intervalb | |
---|---|---|
4 weeks | 8 weeks | |
Chitosan | 0.64 to 1.75 (mean = 1.2) | 0.91 to 2.28 (mean = 1.6) |
Chitosan + Simvastatin | 0.98 to 2.28 (mean = 1.6) | 1.12 to 2.20 (mean = 1.6) |
Regarding degradation, membranes in the control and experimental groups presented with minimal to no change of shape and structure at 4 weeks (
Histomorphometric measurements of percent of membrane in defect area are shown in
As shown in
Chitosan | Chitosan + Simvastatin | ||||
---|---|---|---|---|---|
4 weeks | 8 weeks | 4 weeks | 8 weeks | ||
Histomorphometric measurements M ± SD | % new bone/total defect area | 20.87 ± 13.86 | 32.96 ± 31.61 | 10.08 ± 9.87 | 33.02 ± 33.56 |
% membrane/defect area | 24.79 ± 10.72 | 18.69 ± 13.37 | 25.07 ± 22.86 | 14.90 ± 17.07 | |
Micro-CT analysis M ± SD | % new bone volume/defect volume | 9. 40 ± 6.21 | 28.89 ± 26.90 | 10.63 ± 9.98 | 12.83 ± 4.80 |
density (g/ml) | 1.61 ± 0.05 | 1.69 ± 0.14 | 1.62 ± 0.04 | 1.66 ± 0.02 | |
new bone surface area/new bone volume (mm−1) | 23.19 ± 5.40 | 20.58 ± 15.65 | 26.88 ± 9.21 | 19.37 ± 3.60 |
the defect was higher in the control group (20.87 ± 13.86) compared to experimental group (10.08 ± 9.87), though not statistically significant (P > 0.05). New bone formation increased at 8 weeks for both groups and was slightly higher in the experimental group (33.02 ± 33.56) vs (32.96 ± 31.61) in the control. However, the differences were not statistically significant (P > 0.05).
Micro CT analysis showed apparent areas of bone formation in previously created defects indicating new bone formation at 4 weeks in both groups (
GBR membranes are used to enable predictable bone formation in alveolar bone. The principle characteristics of these membranes are cell occlusivity, to prevent migration of faster growing soft tissue into regenerating bone spaces, biocompatibility to not interfere with bone healing/regeneration processes and clinical manageability to enable membrane adaptation to individual defect shape. Biocompatibility is considered one of the most essential properties of regenerative membranes. Numerous in vitro and in vivo studies have shown electrospun
chitosan nanofiber membranes to exhibit favorable biocompatibility properties by exhibiting normal healing and low inflammatory responses [
The results from this study are in agreement with previous studies citing the general biocompatibility of chitosan electrospun membranes [
Chitosan membranes loaded with simvastatin also exhibited minimal inflammation in our study, with good level of biocompatibility during the healing process. Previous studies have reported that local delivery of optimal dose of simvastatin had no considerable effect on inflammatory and tissue response at the targeted site [
Bioresorbable membranes provide a significant advantage over non resorbable membranes by eliminating the need for a reentry procedure intended for membrane removal. However, early degradation of resorbable membranes may pose a risk of losing the space for bone formation. Therefore, resorbable membranes need to retain their barrier function and maintain the space during the healing period. In this study, clinical observations at retrieval were consistent with the histologic evaluations revealing that both control and simvastatin loaded chitosan membranes remained intact with no significant signs of fragmentation or degradation up to 8 weeks. Even though soft tissue was in close proximity/apposition to both membrane groups, the membranes maintained their barrier function and were effective in preventing soft tissue penetration. Similar histologic observations and measurements for the amount of membrane remaining in both groups suggest that simvastatin has a negligible effect on chitosan membrane rate of degradation.
The slow degradation of the membranes was judged to be appropriate since the membranes should last in place without degradation for 4 - 6 weeks [
In the current study, histologic and micro-CT evaluations confirmed bone formation in calvarial defects as early as 4 weeks. Initially, bony islands were seen below the membranes and thicker osseous tissue was detected at the edges of the defect. Micro-CT analysis demonstrated scattered radiopaque areas of bone formation with comparable results in both groups for all measurements. The results of early bone formation in our study are consistent with previous reports where bone formation was observed in defects at 3 - 4 week time points following surgery [
Bone continued to form in the calvarial defects in both groups at 8 weeks and bridges of newly-formed bone fusing margins of defects were observed histologically. Both membranes appeared to maintain an effective barrier against soft tissue penetration allowing more healing time for localized areas of osseous tissue to connect and thicken. Bone formation was also found within the folded portions of membranes in both groups. Membrane folding can be attributed to animal movements and rubbing in the cage. Histologic observations of bone formation were quantified through histomorphometric measurements showing an increase in bone formation at 8 weeks in comparison to the 4-week time point for both groups with no statistical significance.
Bone formation was observed in a close proximity to membrane surface with no fibrous tissue separation. This histological finding may suggest high affinity of osteoprogenitor cells to chitosan membrane surface which may play an important role in the enhancement of osteogenesis. Previous studies reported an inhibitory effect of chitosan against fibroblasts while stimulating osteoblastic activity [
Micro-CT analysis revealed increased levels of new bone volume and density throughout the study period. The increase in density may be due either to new bone formed at a later stage or new bone formed earlier that eventually mineralized. The control group demonstrated higher values at 8 weeks in comparison with the experimental group. The micro-CT results are not in conflict since each result showed non-significant differences between the two groups. This can be due to the large standard deviation of the new bone volume of the control group.
Although, micro-CT analysis provided a widespread assessment of bone formation at different time points, only mineralized new bone in defect was detected. On the other hand, histomorphometry evaluated bone growth at the mid-sagittal section of the defect regardless of the mineralization status. As a result, less mineralized newly formed bone, that can be observed histologically, may be undetected on micro-CT. This may provide an explanation of lower micro-CT values of bone formation in comparison to histomorphometric values, particularly at early stages of bone formation.
The micro-CT bone surface to volume ratio is considered a good indicator of the active remodeling process within the bony defect. Higher bone remodeling may lead to greater bone surface with healing progression. The decrease in surface area-to-volume of new bone at 8 weeks may indicate increased bone formation in the defect along with a fair amount of osteoid remolding activity. A study by our group reported new bone volume/defect volume percentages of 37.2% ± 22.7% and 13.6% ± 7.1% calculated from histomorphometric and micro-CT data respectively for the butyric acid modified chitosan nanofibrous membranes at 12 weeks [
A major aim of this study was to evaluate whether the local delivery of simvastatin from the chitosan membranes may enhance the new bone formation and defect fill. The successful use of simvastatin to promote bone formation depends on the local effective concentration at the defect by using an appropriate delivery system. Numerous studies have reported that the local delivery of simvastatin from carriers such as α-tricalcium phosphate and polylactic acid membranes lead to an increase in bone healing [
Additional studies are needed to determine the release profile of simvastatin from the chitosan membranes used in our study, as well as the ideal release profile for a beneficial effect on bone healing. The overall results should be taken with caution considering the large standard deviation of the new bone growth that was observed in both groups. Some samples yielded significant new bone formation that almost covered all the defect area, while some only showed minimal new bone even after 8 weeks. Nevertheless, results from this study suggest that chitosan nanofiber membranes may be suitable carriers for simvastatin and a viable alternative to currently utilized GBR membranes.
The present study tested a novel chitosan carrier system to locally deliver simvastatin to promote bone formation. Both unloaded and loaded chitosan membranes were biocompatible, with enhanced bone formation and no evidence of an adverse inflammatory reaction. Slight improved bone formation was observed with simvastatin loaded chitosan membranes, however, more studies are needed to optimize this delivery system and the kinetics of release. This study shows that biodegradable nanofiber chitosan membranes with and without simvastatin may be of a promising potential in the field of guided bone regeneration.
This study was funded by the University of Tennessee, College of Dentistry Alumni Fund and the College of Dentistry Unrestricted Educational Funds. Research was also supported by the Biomaterials Applications of Memphis (BAM) Research Laboratories in the Biomedical Engineering Department at the University of Memphis, Memphis, TN.
Ghadri, N., Anderson, K.M., Adatrow, P., Stein, S.H., Su, H.J., Garcia-Godoy, F., Karydis, A. and Bumgardner, J.D. (2018) Evaluation of Bone Regeneration of Simvastatin Loaded Chitosan Nanofiber Membranes in Rodent Calvarial Defects. Journal of Biomaterials and Nanobiotechnology, 9, 210-231. https://doi.org/10.4236/jbnb.2018.92012