The aim of the present in vivo study is to histologically evaluate and compare the use of resorbable screws based on poly(L-co-D,L lactide) 70:30 for fixation of autogenous bone grafts in rabbit tibiae. As control group, titanium (Ti-6Al-4V Grade V) screws were used. For this purpose, 15 white New Zealand male rabbits, aged 6 months and weighing between 3.8 and 4.5 kg, were used. From each animal, 2 total-thickness bone grafts were removed from the cranial vault: one was stabilized with a resorbable screw while the other was stabilized with a metallic one. Animals were divided into 3 groups, according to the sacrifice period: 3, 8, and 16 weeks postoperatively. After histological processing, cuts were stained with hematoxylin and eosin and submitted for descriptive histological analysis under light microscopy. It was found that the fixation system based on the polymer showed a histological behavior similar to metallic screws. For both groups, the bone graft was incorporated, with the presence of bone formation between the graft and receptor site. In none of the groups were undesirable inflammatory responses or foreign body reactions observed. Based on histological findings and on this experimental model, it is possible to conclude that the internal fixation system based on the poly(L-co-D,L lactide) 70:30 polymer is effective for fixation of autogenous bone grafts, with results that are comparable to the titanium fixation system.

Introduction

Surgical procedures for reconstruction of alveolar ridges became particularly common in the 1990s because of the increased use of dental implants to replace missing teeth. By restoring the natural form and volume of the alveolar ridge, the surgeon is able to place dental implants in an ideal esthetic and functional tridimensional position.1  Other advantages of this procedure include the installation of an increased number of implants, with greater diameters and lengths and better distribution in the arches.2,3 

Various augmentation techniques can be used to provide sufficient bone volume for reliable placement of endosseous implants in the severely resorbed alveolar ridge.4,5  The current gold standard in alveolar ridge augmentation is autogenous bone grafting, particularly block grafts, which have been shown to provide a predictable increase in bone volume for maxillary and mandibular alveolar augmentation.6,7  Confirming the high efficacy of this type of surgical procedure, Triplett and Schow8  demonstrated that implants inserted in augmented ridges with corticocancellous block grafts have a success rate ranging from 85% to 98%.

Once onlay grafting has been chosen as the modality for ridge augmentation, it is imperative that the block graft is properly adapted to the recipient site and remains firmly attached until its incorporation. The results of a study published by Philips and Rahn9  clearly demonstrated increased graft survival rates in rigidly fixated onlay bone grafts when compared with grafts that were not rigidly fixated. La Trenta et al,10  using a dog model, showed an increased survival rate of bone grafts rigidly fixated when compared with those not immobilized. The authors found that the nonfixed grafts predominately healed with a fibrous union. Given these findings, the following statements have been postulated: the immobilization leads to a faster process of revascularization: (1) the compression of the graft with the recipient site increases the contact area between them, resulting in (2) a small resorption phase, (3) earlier onset of the appositional phase of the bone healing cascade, and (4) earlier graft consolidation allowing for early osteogenic cell ingrowth.

Traditionally, metallic screws have been used to achieve rigid fixation for onlay grafting. Despite some limitations, titanium is widely accepted as the metal with the best biocompatibility and excellent mechanical properties and therefore has been successfully used to provide rigid fixation.8,11  Therefore, many authors have recommended the removal of titanium osteosynthesis material after bone healing. Kim et al,12  in light of their findings of local macroscopic and microscopic tissue damage around the implanted titanium plates, proposed that all titanium plates should be removed routinely after bone healing. Potential drawbacks of using titanium plates and screws for alveolar ridge reconstruction include (1) need for removal before implant placement, (2) risk of screw fracture during removal, (3) radiographic artifact during imaging, (4) patient discomfort due to palpability through the oral mucosa, (5) bone atrophy or osteopenia caused by stress shielding and corrosion, (6) allergic reactions, (7) infection, (8) thermal sensitivity, and (9) the possibility of causing growth restriction of the craniofacial skeleton in pediatric patients.1317 

To overcome the mentioned drawbacks of the metallic devices, alternatives to this material were completely investigated, and the number of resorbable fixation systems was increased.1820  The fixation devices consist of a combination of different bioresorbable polymers including polylactide, polyglycolide, and their copolymers,21  so as to achieve a balance between mechanical strength, flexibility, inflammatory response, and resorption time.22  Currently, most devices are manufactured by combining 2 polymers, mainly poly-D-lactide or polyglycolide with poly-L-lactide. This combination reduces the crystallinity of the material, which leads to a very slow resorption and decreased clinically detectable adverse reactions.23 

Although various investigators have demonstrated favorable results using resorbable fixation, the lack of studies using such material in bone reconstruction surgery is evident. The aim of this study was to histologically determine and compare the suitability of poly(L-co-D,L lactide) 70:30 fixation screws for onlay autogenous block graft fixation in rabbit tibiae.

Materials and Methods

Fifteen adult New Zealand male rabbits, aged approximately 6 months and weighing between 3.8 and 4.5 kg, were enrolled the study sample. Thirty minutes before anesthesia, the animals received an intramuscular injection of 0.2 mL/kg of broad-spectrum prophylactic antibiotic. Surgical procedures were performed under general anesthesia using intramuscular injection of ketamine 30 mg/kg and xylazine 6 mg/kg. Prior to surgical incisions, 0.6 mL of 2% lidocaine with 1:100 000 diluted epinephrine solution was administered via local infiltration to induce anesthesia and hemostasis.

The procedure started with access to the recipient site: the medial tibia plateau. A linear incision was performed through the skin and subcutaneous tissue. After dissection and retraction of the muscle, the underlying fascia and periostium were incised, providing adequate access to the posterior-medial tibia (Figure 1a). Next, the donor site was accessed and grafts were harvested from the calvarial bone (Figure 1b). Harvesting of the grafts followed a protocol published by Alberius in 1989.24  A 4-cm linear incision through skin, subcutaneous tissue, and periostium was performed in a layered fashion on each parietal bone. Once the periostium was dissected free and the tissues were reflected as full-thickness flaps, a trephine bur 8 mm in diameter was used to harvest a cylinder block graft from each parietal bone (Figure 1c). Immediately after harvesting, the grafts were perforated in their central portion with a 2-mm perforation bur and then adapted to the recipient site previously exposed. A minimum distance of 4 mm between the grafts was kept. Next, a twist drill 1.6 mm in diameter was used through the 2.0-mm perforation in the center of the graft to perforate the recipient bone. In the control group, the grafts were fixated with 2.0 mm outer diameter and 12-mm-long titanium screws (Ti-6Al-4V/Grau V; Figure 2a), which were inserted directly to the bone through the perforations. In the study group, the grafts were fixated using 2.0-mm outer diameter and 12-mm-long absorbable screws manufactured from an amorphous copolymer derived from lactic acid, poly(L-co-D, L-lactic acid) 70:30 (Figure 2b), which were inserted through the perforations after tapping the recipient bone. Surgical wounds were then sutured in a layered fashion with 3-0 polyglactin 910 and 4-0 nylon suture. The animals were then allowed to recover. Before sacrificing, the animals were randomly divided into 3 groups according to the sacrificing periods 3, 8, and 16 weeks postoperatively. Therefore, each group comprised 5 animals.

Animals were euthanized by intramuscular injection of ketamine 150 mg and xylazine 30 mg, followed by a lethal dose of 300 mg of ketamine administered intravenously. Next, the animals had the operated tibia disarticulated, and the grafts-native bone region was separated from the rest of the tibia using a 701 fissure bur and leaving a 5-mm margin of bone to prevent graft damage. The excised samples were then separated, labeled, and fixed in buffered 10% formalin. After a fixation period of 3 weeks, the specimens were decalcified in a solution of 50% formic acid and 20% sodium citrate for 60 days, replacing the solution every 48 hours. After the decalcification process, the titanium and absorbable screws were removed from the specimens using specific keys.

Clinically, the bone graft was incorporated into the recipient bone in all samples. After removal of the fixation screws, the pieces were dehydrated by immersing in a solution of 50%, 70%, 90%, and 96% of ethyl alcohol and afterward in absolute alcohol. The pieces were kept in ethyl alcohol for 3 hours in each concentration and for 24 hours in absolute alcohol. The pieces were next placed in xylene for 2 hours and then embedded in paraffin for sectioning. Sectioning of the blocks was performed so that a 4-μm longitudinal section of the central portion of the screws was obtained. Sections were then stained with hematoxylin-eosin and examined under routine light microscopy for bone graft incorporation and the contact between the screws and recipient bone. Data were analyzed by basic descriptive analysis.

Results

All animals tolerated the surgical procedure under general anesthesia, and no sample loss occurred in the postoperative period. Gross examination revealed that both groups showed graft consolidation with obvious osseous thickening in the recipient area. There was no evidence of graft mobility. In addition, the surrounding tissues appeared healthy without signs of foreign body reaction or inflammation.

3-week histological analysis

The control group showed fibrous connective tissue at the interface between the bone graft and the recipient bone, although immature bone could be seen forming toward the recipient site. In these same samples, there was neutrophilic infiltration at the interface (Figure 3a). Chambers of the screws predominantly filled with very immature newly formed bone with large vascular channels could be observed in all animals (Figure 3b).

In addition, in the study group, there was deposition of fibrous tissue between the bone fragments. Moreover, in all animals, there was necrosis and more intense neutrophil infiltration when compared with the control group (Figure 4a). Several fragments of irregular and refractile bone and a mild amount of mast cells were present as well. This moderate inflammatory reaction may be responsible for the delayed bone formation in the study group at this time frame. In the screw chambers and the recipient bone, there was also an amount of immature bone formation, but at this time frame, this woven bone was not in contact with the screws threads, as observed in the control group (Figure 4b).

8-week histological analysis

In all animals of the control group, there was more mature bone bridging the graft and recipient site, demonstrating the incorporation process of the graft. This newly formed bone was already characterized with the presence of large channels and osteocytes (Figure 5a). The chambers between the threads of the metallic screws were filled with fibrous connective tissue and, in turn, newly formed mature bone (Figure 5b). There was also inflammatory cell infiltration into the medullary adipose tissue.

The study group followed a similar pattern of graft integration. In 4 animals, the graft showed a good incorporation process into the recipient site. Both tissues were bridged together by newly formed mature bone and a very small amount of fibrous connective tissue (Figure 6a). In 1 animal, there was moderate neutrophil infiltration between the adipocytes in the bone marrow along with multiple irregular fragments of bone. Furthermore, at this time frame, the study group showed a narrow band of newly formed bone in the perimeter of the screws, including the chambers between the threads (Figure 6b).

16-week histological analysis

In all animals of the control group, there was a thick band of lamellar bone between the grafted bone and the recipient site. This bone is characterized by well-organized bone features as medullary spaces filled with blood vessels, osteocytes, and Haversian system (Figure 7a). Newly formed bone can also be appreciated in the chambers between the threads of the titanium screws (Figure 7b).

All samples of the study group at 16 weeks showed a small rupture line between the native and formed bone at the interface graft-recipient site. Some refractile fragments were still involved by adipocytes in bone marrow (Figure 8a). When examining the interface screw-native bone, there was disordered arrangement of bone tissue in the valleys regions. In general, there was a thin band of bone oriented parallel to the medullary canal at the perimeter of the screw, sometimes occurring as Haversian systems (Figure 8b). In some areas, there was a break in the demarcated line between the native and newly formed bone.

Discussion

Scientific and technological development has marked the past 3 decades of advances in dentistry and more specifically in oral surgery. A clear example of all of the improvement achieved is the better understanding of all biological events present in the different stages of bone repair along with the development of new osteosynthesis materials. Undoubtedly, a major breakthrough was the introduction of rigid internal fixation with metal devices. Following the extensive and rapid growth of dental implants, the authors reported the use of such devices for bone fixation in alveolar ridge augmentation procedures.1,7  In particular, metallic plates and screws have become routine for fixation of bone grafts in preimplant surgery.25  However, the use of metallic devices for this purpose offers some disadvantages and potential drawbacks, such as the need for secondary removal before placement of the dental implants, which may be easily performed through a small incision but at times requires more extensive surgical approaches with larger incisions and additional extensive soft-tissue stripping. Moreover, other complications are related to the use of metallic devices.26,27 

It is well documented in the literature that resorbable fixation materials offer many advantages for osteosynthesis over their metallic counterparts. As mentioned, no removal of the material is needed. The elasticity of these materials is close to that of bone, thus enhancing the stress protection.27  During bone healing, the resorbable material will gradually degrade, allowing physiologic stress to be transferred back to the healing bone. As a result, stress shielding is avoided.28 

In 2004, Chacon et al21  were the first to investigate a resorbable fixation system for autologous onlay bone grafting in an animal model. The study showed that resorbable screws provide excellent graft stability at the graft placement phase and at the graft retrieval stage. It was noted that grafts had consolidated to the mandible without evidence of inflammatory reaction or loss of graft volume. In 2006, Raghoebar et al25  published a clinical report on the application of biodegradable screws to fix bone grafts in a human split-mouth model. They reported that the comparison of the titanium vs resorbable screw revealed no differences with respect to wound healing and histologically no signs of a significant inflammatory response to the poly-DL-lactide acid (PDLLA) material except for the abundance of giant cells. In 2010, Quereshy et al29  published another study using resorbable screws for block graft fixation in humans and showed that poly-L-lactic acid (PLLA) with poly-lactic-co-glycolic acid fixation screws had no negative influence on graft integration and survivability of autogenous onlay bone grafts. In accordance with these 3 studies, the present study showed that the use of the resorbable screws for fixation of the bone grafts was as successful as titanium screws. New bone formation could be seen at the interface between the grafted tissue and the recipient site in all groups. Although fibrous connective tissue formation was predominant at the early postoperative period, especially in the study group, a replacement by hard tissue could be seen at 8 weeks followed by organization in lamellar bone by the 16th week. A similar pattern of bone formation occurred at the interface between the screws and the recipient bone. In addition, in the study group, the formation of bone was shortly delayed, but new bone formation occurred throughout the study.

Resorbable screws induced a stronger inflammatory response compared with titanium screws. This was most evident in histological analysis of 3 and 8 weeks. Most likely, this inflammatory reaction explains the slight delay in the process of incorporation of bone grafts fixed with absorbable screws. Nevertheless, the results after 16 weeks were similar for both groups. This difference in inflammatory reaction did not compromise the incorporation of bone grafts, and the low inflammatory response associated with both groups and its reduction throughout the study indicated that the biologic response to the procedure was favorable for graft consolidation. These data are consistent with previous studies using this material as an osseous fixation device.3032 

Historically, bioresorbable polymers have been associated with host tissue reaction and have led to some difficulty using these materials. However, it is possible to combine different polymers (ie, poly-lactic acid + poly-glycolide) or even associate different forms of the same polymer (ie, poly-DL-lactic acid). Hence, various combinations can be obtained by providing materials with different mechanical properties that are resorbed at different periods of time and cause different amounts of tissue inflammatory response. There are basically two forms of polymers of lactic acid (optically active stereoisomer): PLLA and poly-D, L-lactide (PDLA). The PLLA is characterized by a large amount of crystalline particles, high mechanical strength, and long-term degradation. A study associates this high crystallinity and prolonged periods of resorption with an increased incidence of adverse tissue reactions.23  In contrast, the PDLA has a low resistance and rapid decay.33  The creation of a copolymer poly (L-co-D, L lactic acid) in a proportion of 70:30 seeks to optimize the properties of the compound while retaining excellent mechanical strength, faster degradation rate, and fewer inflammatory reactions. These modern bioabsorbable materials such as a combination of copolymers as the one used in the present study are designed to avoid these reactions and thus have shown great success in preimplant bone reconstruction and other oral and maxillofacial procedures.

The polymer tested allowed bone graft incorporation but was still present at the end of this process. The defects where the screw resorbed in the grafted area were partially filled with bone, and remnants of the resorbable screws were also observed. Because there was no severe inflammatory response to the PDLLA material, it was possible to place dental implants in the healed grafted site, drilling through the remains of the resorbable screws without jeopardizing the outcome of the rehabilitation treatment or implant survival rates.

Given the histological findings observed in the study and control groups and respecting the limitations of this study, it is possible to conclude that (1) the resorbable fixation system tested was effective in promoting the incorporation of bone grafts, (2) the resorbable polymer induces a more intense inflammatory response when compared with the titanium system, (3) there was a slight delay in the incorporation of bone grafts fixed with resorbable screws, and (4) both materials proved to be biocompatible and lacked histological evidence of foreign body reaction.

Abbreviations

     
  • PDLA

    poly-D, L-lactide

  •  
  • PDLLA

    poly-DL-lactide acid

  •  
  • PLLA

    poly-L-lactic acid

Acknowledgments

The authors would like to acknowledge Engimplan Implant Engineering Indústria e Comércio Ltda for providing the fixation devices used in the study. As well, the authors would like to thank CNPQ for providing funds for research development. Funding provided by CNPQ (Conselho Nacional de Pesquisa).

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