Combination of poly L-lactic acid nanofiber scaffold with omentum graft for bone healing in experimental defect in tibia of rabbits

PURPOSE: To investigate the osteoconductive properties and biological performance of Poly L-lactic acid (PLLA) with omentum in bone defects. METHODS: PLLA nanofiber scaffolds were prepared via electrospinning technique. Forty four New Zealand white female rabbits randomly divided into three groups of 18 rabbits each. Created defects in right tibias were filled in group I with omentum, in group II with PLLA nanofiber scaffold and in group III with combination of the omentum and PLLA. The same defects were created in left tibia of all groups but did not receive any treatment (control group). Histological and histomorphometric evaluations were performed at two, four and six weeks after the implantation. RESULTS: Histological changes on all groups along with the time course were scored and statistical analysis showed that the average scores in group III were significantly higher than the other groups. CONCLUSION: Histomorphometric analysis of bone healing was shown to be significantly improved by the combined PLLA with omentum compared with the other groups, suggesting this biomaterial promote the healing of cortical bone, presumably by acting as an osteoconductive scaffold.


Introduction
Acceleration of bone healing in fracture site has always been a major problem.Fresh autologous spongy bone transplantation is considered as the gold standard for bone grafts 1,2 .
As compared with other methods the progenitor cells provided without stimulation of immune system response.But it has some problems, including donor site damage and limitation of harvested amount.These restrictions encourage researchers to use synthetic bone graft substitutes 3,4 .
Matrix-based tissue-engineering approaches aim to use structural implants and/or materials to replace the defective bone 5,6 .These approaches depend on the recruitment of endogenous osteoinductive factors and migration of osteogenic cells to regenerate bone defect 7,8 .If the scaffold could induce cells from the surrounding tissues, pre-expansion of the cells for the transplantation would not be required 9 .After insertion of a material to damage site, it will be exposed to adjacent cells, resulting in captured repair cells and the possibility of leakage and non-homogeneous scatter will be decreased.
However about the use of these materials, complete success has not yet achieved 14,15 because their physical structures are different from bones.Furthermore, they may have osteoconductive but normally not osteoinductive properties and can only act as a matrix for new osseous ingrowth and slow creeping substitution.
The time-consuming process of resorption is also a problem 14 .
PLLA is an organic polymer from L-lactate that dissolves in H 2 O and CO 2 .PLLA was used as a material for human orthopedic implants 19 .In vitro studies showed that PLLA scaffolds had slow absorption rates and good mechanical 20 and biocompatible properties 21 .
Given this, combined omenton with Synthetic bone graft substitutes may improve the bone tissue formation and repair of bone defects.The aim of this study was to investigate the osteoconductive properties and biological performance of PLLA combined with omentum in bone defects.
Electrospinning is one of the approaches that allow the fabrication of synthetic materials into fibrous structures in the micro-and nanometer scale 21 .In this research PLLA nanofiber Scaffolds were prepared via electrospinning technique, and then these biomaterials alone and with omentum were implanted in experimental bone defects.Histological and histomorphmetric evaluations were performed at two, four and six weeks after the implantation.

Methods
All animal of the present research were cared according to the norms of the Islamic Azad University Faculty of Veterinary Sciences laboratory of animal experimentations; this investigation was approved by the Committee of Ethics in Research with animals too.

Scaffold preparation
Nanofibrous PLLA scaffolds were prepared via electrospinning technique.A 4 % (wt/wt) solution of PLLA (Sigma-Aldrich, St. Louis, MO, USA) in dichloromethane/ dimethylformamide (Merck, Germany) was placed in a 5 mL syringe which was connected to a blunted 21-gauge needle through an extension tube.A steel grounded collector was used to collect the electrospun nanofibers.The solution was fed through the tube into the needle by a syringe pump with a rate of 0.5 mL/h.Application of high voltage (20 kV) between the needle and collector, forced the solution droplet to leave the needle and deposit on the cylinder in the form of nanofibers.In order to modify surface characteristics of prepared scaffolds, surface plasma treatment was performed by a low frequency plasma generator of 40 kHz frequency with a cylindrical quartz reactor (Diener Electronics, Germany).Pure oxygen was introduced into the reaction chamber at 0.4 mbar pressure and then the glow discharge was ignited for three min.

Scanning electron microscopy
The surface morphology of scaffolds was characterized using a scanning electron microscope (SEM, Philips XL30, Netherlands) after specimens were coated with gold using a sputter coater.

Scaffold implantation
Forty four New Zealand white adult female rabbits were used with following ethical approval as previously reported.The rabbit's age was eight months and the weight 3000-3500g.
They were randomly divided into three groups of 18 rabbits each.
The rabbits were anesthetized with an intramuscular injection of ketamine hydrochloride (30 mg/Kg of body weight) and xylazine (5 mg/Kg of body weight).The animals were immobilized, and the bilateral tibias were shaved, washed and disinfected with povidone-iodine.A 4 cm incision was made on the proximalanterior part of tibiae.The incision penetrated epidermis, dermis and the fascial layers.An additional medial-anterior incision was made through the periosteum.The periosteum was elevated with a periosteal elevator and retained by a selfretaining retractor.A cortical hole of 3mm diameter and depth in each tibia was drilled.
The bone cavities were washed with saline during and after the drilling.
Additionaly in group I and III, 3*3 mm 2 of the free end of the greater omentum obtained from a 1 cm ventral midline incision midway between the umbilicus and pelvic inlet.Created defects in right tibias were filled in group I with omentum, in group II with PLLA and in group III with combination of the omentum and PLLA.The same defects were created in left tibia of all groups but did not receive any treatment and will be considered as control group.After implantation, Muscles, fascia and skin were sutured separately.
To reduce the perioperative infection risk, prophylactic antibiotic (Terramycines) was administered postoperatively by subcutaneous injection.The animals received a postoperative pain medication and were allowed to move without any restrictions immediately after recovery from the anesthesia.

Histological analysis
Six of the animals in each group were respectively sacrificed at the end of two, four and six weeks.The right and left tibias were harvested at the designated time points.The specimens were harvested with the surrounding tissue and fixed in 10% neutral formalin and were decalcified with 10% nitric acid, dehydrated, and then embedded in paraffin, after that were sectioned at 5 µm and stained with Hemotoxylin-eosin for histological observation (Table 1).Assessment was performed by a blinded assessor while considering parameters in table 1, with an Olympus BH-2 light microscope (Tokyo, Japan).

Histomorphometric analysis
Measurements were made on two sections for each sample.
All sections of specimens were used for histomorphometrical evaluation using computer based image analysis techniques (Leicas Qwin Pro-image analysis system, Wetzlar, Germany).
Digital analysis determined the total bone formation (%).This parameter was subdivided in: (A) bone inside the implant (BII), defined as bone formed within the implant edges; (B) bone outside the implant (BOI) defined as newly formed bone deposited outside the implant.

Statistical analysis
Analysis of variance (ANOVA) was used to evaluate the differences between groups as well as between different time points in a group.Statistical analysis was performed using the SPSS statistics package version 15.0.15.

Characterization of nanofibers
SEM micrographs of PLLA scaffolds are shown in  Combination of poly L-lactic acid nanofiber scaffold with omentum graft for bone healing in experimental defect in tibia of rabbits Acta Cirúrgica Brasileira -Vol.27 (10) 2012 -697

Histological evaluations
After two weeks In Group I, there was very little inflammation and new bone formation was already.Contact with bone graft material was also seen, Figure 2A.In group II, no evidence of foreign body response to the PLLA was present but the inflammation in this group was remarkable, Figure 2B.In Group III newly formed woven bone (BII) early appeared and bone bonding between the implants and host bone were observed, Figure 2C.

After four weeks
The woven bone was formated in group I and partial direct contact between the trabeculae and the garft occurred, Figure 3A.Group II was also observed woven bone formation and there was a little inflammation in this group.Bone growth was BOI and BII but contact with the host bone was not well.No macrophages were found around the implants, Figure 3B.Mixed lamellar and woven bone formation was seen in group III and inflammation was very low compared to the second week.Foreign body reaction was negative.Bone growth was observed as well as the BOI and BII.
Bone lacunas contained osteocyte that demonstrated bone tissue was active and vital, Figure 3C.Detailed results of Histological evaluations are summarized in Table 2. Whereas Histological changes on all groups along with the time course were scored and statistical analysis showed that the total average scores in group III were significantly higher than the other groups (p<0.05).

Histomorphometrical evaluations
The results of the histomorphometrical analysis are shown in Table 3.In all four groups the formation of new bone increased all the time periods.Comparison between groups I and II showed no significantly difference in formation of new bone after two, four and ten weeks (p<0.05).
After two weeks in the group III, formation of new bone was 25.75% (BII: 25.75%; BOI: 0.0%).After four weeks it was 36.75% (BII: 29.14%; BOI: 7.61%) and it increased to 42.53 (BII: 32.44%; BOI: 10.09%) after 6 weeks.Statistical testing revealed that these increases were significantly different in formation of new bone after two, four and ten weeks (p<0.05)compared with other groups.

Discussion
Biomaterial scaffolds, osteogenic cells, and bone formation-inductive factors are three essential elements for tissue engineering strategies to achieve repair and restoration of damaged bone tissues [37][38][39][40][41][42] .Scaffolds are central components of many tissue engineering strategies and there is a significant challenge in the design and manufacture of scaffolds that possess both a highly porous structure and the ability to control the release kinetics of growth factors over the period of tissue regeneration 43 .
An ideal biomaterial for bone tissue engineering should be nonimmunogenic, highly effective in osteoinduction, ready for rapid vascular and mesenchymal cell invasion, carvable and providing mechanical support when needed 43 .
Various types of biomaterials have been utilized as Scaffold.Currently, bioactive glasses in the form of granules are used to enhance bone healing.However, the clinical handling properties of them are limited 45 .Bone cements have been shown to conduct bone growth, but will also induce foreign-body cell reactions 46 .Poor mechanical strength and insufficient adhesion between the bone and the cement as well as low degradation rate restricting the use of some bone filler materials 47,48 .Metals are difficult to shape and have a problem of corrosion which leads to inflammatory responses of surrounding tissue 49,50 .
Scaffold porosity and interconnections or pathways between the pores are vital for new bone integration into the material 51 .The high ratio of surface to volume and high porosity of three dimensional nanofibers scaffolds support binding and migration of mesenchymal stem cells into the scaffold that mainly due to the chemical and morphological similarities of them to natural extracellular matrix.These scaffolds readily allowed invasion of the requisite proteins and cells for bone formation and resorption 52 and can support growth and differentiation of MSC 53,54 in vitro as well as in vivo 55 .
The purpose of this study was to evaluate a short term bone response to nanofiber scaffold of PLLA with combination of omentum.
In this study, the surface plasma treatment was used to improve the hydrophilicity of prepared hydrophobic PLLA nanofibers.Oxygen plasma treatment introduces oxygen containing functional groups on the surface of nanofibers which leads to higher hydrophilicity.Increased hydrophilicity facilitates cell adhesion and proliferation on the scaffold surface 56 .
PLLA is a biodegradable semi-crystalline polymer derived from lactic acid (C3H6O3).The main advantages of these polymers are their mechanical strength, combined with biodegradability and biocompatibility 57 .In a similar study, PLLA nanofibers scaffolds with growth factors were examined in the calvariom bone defect and results showed that scaffold had bone Combination of poly L-lactic acid nanofiber scaffold with omentum graft for bone healing in experimental defect in tibia of rabbits Acta Cirúrgica Brasileira -Vol.27 (10) 2012 -699 conductivity properties and also induced osteoinduction 58 .
Electrospun hydroxyapatite-PLLA scaffold was evaluated on sternal bone healing and histological evaluation demonstrated presence of newly formed bone trabeculae among scaffold fibers 21 .In the same manner PLLA nanofiber scaffolds were shown to facilitate cell immigration and thus to achieve high cell densities and the histological evaluations showing an improved healing and tissue remodeling higher degree of maturity in the simultaneous application of PLLA nanofiber scaffold and omentum, with respect to the other group specially six weeks after implantation that suggesting this biomaterial combination has bone conduction properties.The most successful use of this material maybe due to bone regeneration stimulation of growth factors of omentum.Olumi et al. 59 reported that autogenous free omental graft accelerated collagen synthesis in experimental tibial defects in rabbit.In another study the greater omentum were used for femoral defects healing in rabbits and histological results showed bone formation process were faster than the control group.
In this study we demonstrated the use of this scaffold in weight bearing area and similar results were seen.The short 6-week follow-up period was a limitation of this study.However, the aim of this study was to evaluate early histological changes, long-term biocompatibility of the composite needs to be evaluated in a separate study.

Conclusions
The omentum and PLLA alone had little effects on the formation of new bone as demonstrated by histomorphometry but their results were better than control group.Histomorphological analysis of the bone healing was shown to be significantly improved by the combined PLLA with omentum compared with the other groups, suggesting that this biomaterial promote the healing of cortical bone, presumably by acting as an osteoconductive scaffold.

Figure 1 .
Figure 1.The results indicate that fabricated PLLA scaffolds have highly porous structure with interconnected pores and beadfree nanofibers.An ideal scaffold should mimic the physical and biochemical properties of natural extracellular matrix like good porosity and nano-sized structure 1 .As can be seen in the SEM micrographs, electrospun PLLA nanofibers have porous and nanoscale structure so have the potential to be used as scaffold in tissue engineering.

FIGURE 1 -
FIGURE 1 -SEM micrographs showed highly porous structure with interconnected pores and bead-free nanofibers.

FIGURE 2 -
FIGURE 2 -A.In group I after two weeks, omentum graft and immature new bone formation is evident.B. In the group II ostebelastic rim and direct bone contact with biomaterial and new bone formation is evident.C. In group II new bone formation is evident (magnification 10×10, H&E staining.NB=New Bone, I=Inflammation, OM=Omentum).

FIGURE 3 -
FIGURE 3 -A.In group I after four weeks, omentum graft and bone formation is evident.B. In group II PLLA and bone formation is evident.C. In group III bone formation is evident (magnification 10×10, Islamic Azad University (IAU), Kahnooj Branch.H&E staining, NB=New Bone, I=Inflammation, OM=Omentum).

FIGURE 4 .
FIGURE 4. A: In group I after 6 weeks, omentum graft and bone formation and direct contact of bone with graft material is obvious.B: In group II PLLA and bone formation is obvious.C: In group III bone formation is obvious.(Magnification 4×10, H&E staining, OM=Omentum, W=Woven Bone, L=Laminar Bone).

TABLE 2 -
Bone repair histological scores.group III is significantly different from another groups in all weeks, p<0.05. *
*group III is significantly different from another groups in all weeks, p<0.05.