Abstract

This study aimed to investigate and compare the effect of chitosan sponge and platelet-rich plasma (PRP) gel alone as well as their combination on bone regeneration in rabbit cranial defects. Four cranial defects with a 4.5-mm diameter were created in rabbit cranium and grafted with PRP, chitosan sponge alone, and chitosan sponge incorporated with PRP. The rabbits were killed by the fourth and eighth weeks, and the defects were analyzed histologically. Higher bone formation was observed in the PRP group when compared with the other groups at weeks 4 and 8. All parts of the defects were filled with thick trabecular new bone in the PRP group. The amount of new bone formation in the control groups was found to be less when compared with the PRP group and the least in the chitosan group. The defects that were filled with chitosan sponge showed a limited amount of new bone formation and an obvious fibrous demarcation line between chitosan particles and bone. Application of PRP showed a histological tendency toward increased bone formation. Other forms or derivatives of chitosan may have beneficial effects to achieve new bone regeneration.

Introduction

Guided bone regeneration is an accepted surgical procedure intended to increase the quantity and quality of host bone in localized defects of the alveolar bone. Methods described to increase the rate of bone formation and to augment the bone quantity include the use of autografts, allografts, xenografts, and alloplastic bone substitutes.1 

The key to tissue engineering is stimulating a series of events and cascades at a point, which can result in the coordination and completion of integrated tissue formation. Various biological approaches have been used for the promotion of bone regeneration, such as the use of growth and differentiation factors, application of extracellular matrix proteins, attachment factors, and use of mediators of bone metabolism.2 

Polypeptide growth factors are biological mediators that have the ability to regulate cell proliferation, chemotaxis, and differentiation. Although many natural or recombinant growth factors are introduced to increase bone formation, their clinical use is limited because of the cost and immunological problems. Their short life and inefficient delivery to target cells are major concerns.3 Another easy, cost-effective way to obtain high concentrations of growth factors for tissue healing and regeneration is platelet-rich plasma (PRP). Platelet-rich plasma is an autologous concentration of human platelets in a small volume of plasma. It is also a concentration of the 7 fundamental protein growth factors including platelet-derived growth factor (PDGF) αα, PDGF ββ, PDGF αβ, transforming growth factor (TGF) β1, TGF β2, vascular endothelial growth factor, and epithelial growth factor. It also contains cell adhesion molecules such as fibrin, fibronectin, and vitronectin.4 Although these growth factors are known to enhance regeneration, there is still a great need to develop a formable and osteoconductive scaffold for tissue regeneration that would also serve as a vehicle for matrix components and growth factors.

Recently, the use of chitosan in bone regeneration has gained particular interest. Chitosan is partially N-deacetylated chitin. Chitin is a linear homopolymer of 1.4-β linked N-acetyl-D-glucosamine, which is obtained from chitin-rich crab shell. It has been reported to be nontoxic and bioresorbable when used in human and animal models.5 Chitosan can be used to inhibit fibroplasias in wound healing and to promote tissue growth and differentiation.6 Chitosan provides a nonprotein matrix for 3-dimensional tissue growth.7 In addition, chitosan has been shown to regulate the release of bioactive agents including growth factors.8,9 It has been shown to enhance bone formation in vitro and in vivo.10,11 Favorable biological properties of chitosan and its availability in various forms including powder, gel, and solution make it a good candidate for clinical applications as a carrier in periodontal and bone regeneration. Bone-forming materials such as bone powder and synthetic polymers have demonstrated effective bone regenerative potency.12,13 However, drawbacks of these materials may include disease transmission, inappropriate biodegradation, immune response, low tissue compatibility, and poor adaptation.13,14 A significant benefit in using chitosan may be that its degradation product is neutral to weak base sugars, and the superior tissue compatibility of chitosan may primarily be attributed to its structural similarity to glycosaminoglycan in extracellular matrix.

Chitosan may be an effective carrier system for growth factors. Yong-Moo Lee and coworkers showed effective therapeutic concentrations of PDGF-BB released from chitosan sponges up to 21 days after initial burst.15 It has been shown that chitosan is a useful tool to deliver other growth factors including basic fibroblast growth factor16 and TGF β1.7 

Reports regarding the effects of the combination of chitosan with PRP showed an increase in the release of growth factors from PRP8,9 and also increased glycoprotein IIIa expression in platelets.9 Because of the observations of growth factors releasing from activated human platelets after chitosan stimulation, it has been suggested that chitosan can be an appropriate substitute for thrombin in PRP preparation.9 However, the effect of the combination of these 2 agents on bone regeneration still remains unclear.

The aim of this study was to investigate and compare the effect of chitosan and PRP alone as well as their combination on bone regeneration in rabbit cranial defects.

Materials and Methods

Animals and care

The experiments were undertaken on 15 white male New Zealand rabbits weighing between 2500 and 3500 g. The animals were kept in rooms illuminated from 7:00 am to 7:00 pm (12-hour light/dark cycle) that were maintained at 21°C to 23°C, and they had full access to standard dry food and water ad libitum. All animals remained healthy throughout the duration of the study.

Preparations of chitosan sponge and PRP gel

Chitosan with a degree of deacetylation of 86% from FMC Biopolymer (Protasan UP CL 213) was dissolved in distilled water to obtain gel at 3% wt/vol concentration and frozen at −22°C for 24 hours and dried in the lyophilizator to achieve sponge form. The PRP was prepared from blood collected in the immediate preoperative period. Eight milliliters of blood drawn from each rabbit was combined with 1 mL of anticoagulant citrate dextrose phosphate to prevent coagulation. The color-coded PRP kit (Curasan, Pharma Gmbh AG, Lindigstrab, Germany) was used for preparing PRP according to the protocol described previously.17 Briefly, the blood sample was drawn into a citrated tube. The sample tube was then spun in a standard centrifuge for 10 minutes at 2400 rpm to produce platelet-poor plasma. The platelet-poor plasma was taken up into a syringe with a long cannula and an additional air-intake cannula. A second centrifugation (15 minutes at 3600 rpm) was performed to concentrate the platelets. The second supernatant was also taken up by a long cannula and an air-intake cannula. For each 10 mL of blood, the volume of supernatant was about 0.6–0.7 mL: this was the PRP, to be used for the surgical procedure. At the time of application, the PRP was combined with an equal volume of a sterile saline solution containing 10% calcium chloride (a citrate inhibitor that allows the plasma to coagulate) and 0.1 mL of blood that was obtained from the surgical area, including thrombin, which is an activator that allows polymerization of the fibrin into an insoluble gel and causes the platelets to degranulate and release the indicated mediators and cytokines. The result was a sticky gel that can be applied easily to the surgical defects.

Surgical protocol

The rabbits were intubated and underwent general anesthesia with 1% to 2% isoflurane with standard monitoring. The surgical area was prepared by shaving and washing with 7.5% povidone-iodine solution (Poviiodeks scrub, Kim-Pa, Turkey). An appropriate incision was made, and both skin and periosteum were elevated. A 4.5-mm round osteotomy was made in the cranium with a trephine bur with copious irrigation (Figure 1a). Four equal defects were grafted with PRP alone, chitosan sponge alone, and PRP in combination with chitosan sponge, and 1 of the defects was used as control (Figure 1b). All defects were sealed using a nonresorbable 20-mm × 30-mm cytoflex-polytetraflourethylene membrane (Cytoflex, Unicare Biomedical, Inc, Laguna Hills, Calif) to provide initial healing stability of the PRP and chitosan and to prevent tissue infiltration into the defects from their superior apertures. The wound was closed with 4-0 vicryl sutures. Gentamycin suspension (Getamisin, Deva, Turkey) 0.1 mL/kg was given to prevent postoperative infection to the animals. Following surgery, each animal was kept in a 25°C incubator until it regained consciousness. Animals were housed in separate cages.

Tissue processing and histological evaluation

The rabbits were killed using phenobarbital 100 mg/kg by the fourth and eighth weeks. The entire cranium was removed without encroaching on the grafted areas. The specimens were fixed with 4% formalin solution and then decalcificated in 10% formic acid for 48 hours. The specimens were oriented such that both the surrounding healthy bone and the surgical defects could be seen in the same section. The histological specimens were prepared in the usual fashion with hematoxylin-eosin at 5-µm thickness. The histological sections were evaluated using a light microscope.

Results

Histologic results of 4 weeks

Control

New bone formation organized in thin trabeculae covers the whole defect. Numerous blood vessels at the stroma are clearly detectable. Defect surfaces were covered with a thin connective tissue capsule. Active osteoblasts surrounded the newly formed bone. Active stroma between some of the trabeculae was noticed. There was no evidence of inflammatory response at any section (Figure 2a).

Platelet-Rich Plasma

The defects are covered by new bone, and fibrous stroma was detected between the newly formed bone trabeculae at the base of the defects. Osteoblastic activity was abundant even at the superficial layers of the defects. The surgical defects were covered by a connective tissue, which looks like periosteum. Intensive cellular activity and active fibroblastic stroma between the bone trabeculae were notable. There was no evidence of inflammatory response at any sections (Figure 2b).

Chitosan

Eosinophilic-stained chitosan sponges were detectable homogenously surrounded by necrotic cells and inflammatory infiltrate. Although chitosan particles are encapsulated with dense inflammatory regenerated granulation tissue, no cellular activity is detected inside this area. Meanwhile, at the bottom of the defect, the bone covering the existing bone is encircled with the active osteoblasts. New bone with osteocytic lacunae is also observed around this area. There is no evidence of organization of chitosan sponges at this point (Figure 2c).

Chitosan + PRP

Remnants of chitosan were detectable surrounded by inflammatory tissue. New bone formation was observed at the base of the defects. Fibrous connective tissue demarcation without inflammation was detected between chitosan and newly formed bone. Intensive osteoblastic activity was detected around the newly formed bone. Recently formed bone was characterized by the wide lacunae with osteocytes (Figure 2d).

Histologic results of 8 weeks

Control

New bone formation with a thick fibrous border around the defect was observed. Active osteoblastic activity and a fibrous capsule around new bone trabeculae were observed. It was notable that bone formation at highly vascularized sites of the defects was not completed. Neither a necrotic tissue nor an inflammatory process was observed at the defects (Figure 3a).

Platelet-Rich Plasma

Defects were filled by thick bone trabeculae. Limited active connective tissue stroma were notable between bone trabeculae. Absence of osteblastic activity around most of the bone trabeculae implied evidence of the completion of the regeneration. The observation of osteoclastic resorption in some sites might be due to the beginning of the remodeling process. The amorph characteristics of the new bone and the absence of developmental layers may be signs of quick bone formation. Interestingly, in some sites, newly formed bone was so dense that almost no bone marrow could be differentiated (Figure 3b).

Chitosan

Chitosan particles can be detected in the inflammatory infiltrate in the defect. Chitosan particles were less and the fibrous encapsulation become thicker when compared with the sections at the fourth week. Inflammatory infiltrate was detectable inside the fibrous encapsulation between the chitosan and the base of the defect. Newly formed bone was detectable in only a few of the defects. Osteoblastic activity at the areas of appositional bone formation above the former bone was evidence of the unfinished new bone formation (Figure 3c).

Chitosan + PRP

Remnants of Chitosan particles were present in some of the defects. The necrotic tissue among chitosan particles was encapsulated by healthy fibrous tissue. There was no inflammation between chitosan particles and the basement of the defect. This area was covered by fibrous tissue. Osteoblastic activation can still be detectable. In some defects, chitosan particles were completely resorbed, and the remaining chitosan particles were covered with necrotic tissue. Unresorbed chitosan particles were in contact with the newly formed bone. The existence of the fibroblastic activation at the fibrous stroma between the bone trabeculae is evidence of the continuation of the regeneration (Figure 3d).

Discussion

Growth factors are a realistic way to improve and expedite both soft-tissue and bony wound healing. Platelets contain angiogenic, mitogenic, and vascular growth factors in their granules. Platelet-rich plasma is an autologous concentration of platelets in a small volume of plasma. It is a concentration of the 7 fundamental protein growth factors proven to be actively secreted by platelets to initiate all wound healing.4 

The positive effect of PRP on attachment, proliferation, and synthesis of osteoblast was shown in vitro.18,19 Platelet-rich plasma is usually used with the combination of bone graft materials. There are many studies showing that the combination of PRP with bone graft material has enhanced bone regeneration.2025 However, recently there have been some reports conflicting with the results of these previous studies.26,27 Furst et al26 examined bovine hydroxyapatite in combination with PRP for its usefulness for sinus grafting in minipigs and demonstrated that PRP and hydroxyapatite combination is not superior to hydroxyapatite alone. Shanaman and coworkers27 used PRP with demineralized freeze-dried bone allograft for augmentation of alveolar bone and stated that the PRP did not appear to enhance the quality of newly formed bone. Wiltfang and coworkers28 also stated that PRP did not cause additional benefit when xenogenic bone substitutes were used. The addition of PRP hardly influenced bone regeneration, ceramic degradation, or cytokine expression when combined with bone substitutes. However, a significant effect of PRP on bone regeneration was found in the autogenous group with the addition of PRP.28 

In this study, all parts of the defects were filled with thick trabecular new bone in the PRP group. This bone formation was higher when compared with other groups at weeks 4 and 8. These results were similar to the studies of Anitua29 and Akca et al,30 in that the use of PRP alone enhances wound healing and bone formation.

In this study, defects filled with PRP had more bone fill when compared with the control groups. This could be due to either the release of growth factors from PRP or the sticky clot formation that was caused by the use of PRP. Platelet-rich plasma increases mitogenesis and angiogenesis at the beginning of wound healing. On the other hand, osteoconductive graft materials have a longer vascularization period and stay intact for a long time, which causes a limited effect when used in combination with PRP. Use of PRP alone or in combination with autogenous graft materials may have a better effect. There are also many studies showing a significant increase in wound healing and bone remodeling with PRP and the autogenous graft material combination.28,31 

Use of chitosan had limited effects on bone regeneration in this study. Remnants of chitosan particles have been observed in all histological sections. Osteoconductive properties of chitosan can be questionable because of the observation of the fibrous demarcation line between chitosan and newly formed bone at both 2 time points. The defects that were filled with chitosan sponge alone showed a limited amount of new bone formation, which may be explained with inadequate resorption of chitosan.

Susceptibility of chitosan to depolymerization exerted by lysozyme is confined to its degree of deacetylation.5 Tomihata and Ikada32 found that if chitosan is more than 73.3% deacetylated, it may have slower biodegradation. In this study, 86% deacetylated chitosan was used. It has been shown that chitosan that is more than 85% deacetylated can depolymerize in several months.33 The sponge form of chitosan may have caused a delay in degradation. Muzzarelli et al6 have shown that chitosan used in gel form is progressively depolymerized by lysozome and no longer detected at 4 months after insertion in extraction sockets.

In this study, there was an obvious fibrous demarcation line between the bone and chitosan sponge. In contrast, Lee et al34 reported that defects treated with chitosan sponge alone in rat calvaria showed bone growth placed centripetally toward the chitosan matrices. Porous chitosan matrices were embedded in newly formed osseous tissue without fibrous tissue invasion.34 However, chitosan used in this study was 70% deacetylated, and these results may be obtained by the rapid depolymerization of chitosan.

In another study, bone defects were surgically created in the tibiae of rabbits and filled with freeze-dried methylprolidone chitosan. A considerable presence of neoformed bone tissue was observed after 60 days. Inflammatory cells were detected associated with a fibrous mesenchymal tissue, which appeared to be the structural substrate for the neoformation of the osseous trabecule.35 Similarly, in this study, inflammatory cells around unresorbed chitosan particles were observed at the fourth and eighth weeks. This inflammatory process may have also affected the bone regeneration in this group.

Defects filled with chitosan sponge incorporated with PRP gel showed better new bone formation than the chitosan-alone group. This might be due to the effects of growth factors in PRP and activation of macrophages, which are responsible for lysis of chitosan. It is known that growth factors released from PRP activate macrophage chemotaxis and increase lysozomal enzymes released from macrophages.36 Although greater bone formation was seen in this group than the chitosan-alone group, there were still chitosan particles that were encapsulated with fibrous connective tissue.

Recently, Okamoto and coworkers8 evaluated the effects of chitosan on blood coagulation and found that platelets adhered strongly on the surface of chitosan particles with an elongated process. They have also found that chitosan incorporated with PRP enhance the release of PDGF-AB and TGF β1 from platelets.8 One must keep in mind that chitosan used in this study had a deacetylation degree of 10% to 80% and the PRP and chitosan mixture was prepared by centrifugation of PRP and chitosan liquid. Since the chitosan used in this study was in sponge form and had a higher deacetylation degree, expected bone regeneration may have not been achieved.

Theoretically, 3 strategies could be attempted to immobilize bioactive molecules onto carrier material.37 They are covalent and noncovalent linkage of growth factors with specific molecules.38 Such strategies enabled preservation of the bioactivity of growth factors and their sustained release from material substrates.39 Another way to immobilize molecules is the physical entrapment of growth factors into delivery vehicles and release during degradation of the carrier material.40 The third method to immobilize growth factors onto carrier material has been used in this study. As a limitation of this study, the growth factors were not loaded with chitosan sponges and then freezed and dried; they were mixed and inserted in the defects only after obtaining a homogeneous mixture, which does not make it possible to determine the concentrations of growth factors released from sponges.

Based on the present results, it can be concluded that use of PRP in bone regeneration showed a histological tendency toward increased bone formation. However, more investigations should be conducted in different test conditions, especially to determine the time-dependent biological effects of PRP. Use of the chitosan sponge to regenerate bone defects did not improve new bone formation because of the delayed degradation of the sponge. However, the results of the present study do not imply that chitosan cannot be used in bone reconstructive surgery. It is possible that it may function properly if the configuration and the structure of the material are modified.

Abbreviations

PDGF: platelet-derived growth factor

PRP: platelet-rich plasma

TGF: transforming growth factor

Acknowledgments

The authors would like to thank Zafer C. Çehreli, DDS, PhD, for his critical help in preparation of the figures in this article. This study was supported by Gülhane Military Medical Academy Research Center (grant GATA-AR-2003/43).

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Author notes

1

Faculty of Dentistry, Department of Periodontology, Hacettepe University, Sihhiye, Ankara, Turkey.

2

Associate professor, Gülhane Military Medical Academy, Department of Orthopedics and Traumatology, Etlik, Ankara, Turkey.

3

Associate professor, Hacettepe University, Faculty of Dentistry, Department of Periodontology, Sihhiye, Ankara, Turkey.

4

Professor, Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, Sihhiye, Ankara, Turkey.

5

Professor, Hacettepe University, Faculty of Dentistry, Department of Periodontology, Sihhiye, Ankara, Turkey.