Antonietta M Gatti
email: gatti@unimo.it

Leopoldo A Simonetti

Emanuela Monari

Stefano Guidi

David Greenspan

Bone Augmentation with Bioactive Glass in Three Cases of Dental Implant Placement

Keywords: bioactive materials, implants, dentistry, bone defects

This study reports the clinical use of a bioactive bone graft material, PerioGlas Õ , in the treatment of dental extraction sites before dental implant placement, to effect bone regeneration and to give early fixation to the implant.

PerioGlas Õ , granules, ranging from 90 to 710 mm, are implanted after tooth extraction in three patients; after 6 months bone biopsies were performed in the site of the glass implantation and observed under Electron Scanning Microscopy.

All the granules showed a biodegradation involving precipitation of calcium phosphate that worked as a scaffold for osteoblasts colonization. All cases examined showed the bioactivity of PerioGlas Õ granules resulting in new bone formation and biodegradation of the glass. After a two-year clinical follow-up all the implants were successfully loaded and appeared stable.

INTRODUCTION

T he use of dental implants to restore function in partially/ completely edentulous patients has been highly successful in recent years [START_REF] Bra ˚nemark | Ten-year Survival Rates of Fixed Prostheses on Four or Six Implants Ad Modum Branemark in Full Edentulism[END_REF][START_REF] Lekholm | Survival of the Bra ˚nemark Implant in Partially Edentulous Jaws: A 10-year Prospective Multicenter Study[END_REF]. With the development and improvement in dental implant technology, the clinician has more tools to handle complex cases of osseous defects using bone graft materials to augment the sites where the volume of bone would otherwise prohibit the implant placement. There are various materials available for correcting dento-alveolar ridge deformities and augmenting other types of osseous defects. These materials include autogenous bone, demineralized freeze-dried bone allograft (DFDBA), xenografts, alloplasts, and various resorbable and non-resorbable membranes.

Autogenous bone is still considered the gold standard in bone grafting procedures [START_REF] Goldberg | Natural History of Autografts and Allografts[END_REF][START_REF] Becker | Histologic Findings after Implantation and Evaluation of Different Grafting Materials and Titanium Micro Screws into Extraction Sockets: Case Reports[END_REF][START_REF] Becker | Clinical and Histologic Observations of Sites Implanted with Intraoral Autogenous Bone Grafts or Allografts, 15 Human Case Reports[END_REF], notwithstanding its limited availability and the need for a second surgery. The results can be variable, depending on the quality/quantity of the bone used, the preparation of the bone, and the method of placement into the surgical site.

Although there have been reports showing poor results with DFDBA when implanted into extraction sites [START_REF] Becker | Histologic Findings after Implantation and Evaluation of Different Grafting Materials and Titanium Micro Screws into Extraction Sockets: Case Reports[END_REF][START_REF] Froum | Histological Comparison of Healing Extraction Sockets Implanted with Bioactive Glass or Demineralized Freeze-dried Bone Allograft: A Pilot Study[END_REF], it is widely used for bone grafting. Xenografts have also been a popular choice and are said to stimulate bone formation and to be resorbable as well. Recent reports have shown that these materials remain in the implant site for years before resorbing. The persistence of the material in the site over extended time periods could lead to stress concentration and ultimate bone failure [START_REF] Artzi | Porous Bovine Bone Mineral in Healing of Human Extraction Sockets: 2. Histochemical Observations at 9 months[END_REF][START_REF] Hallman | Histologic Analysis of Clinical Biopsies Taken 6 Months and 3 Years after Maxillary Sinus Floor Augmentation with 80% Bovine Hydroxyapatite and 20% Autogenous Bone Mixed with Fibrin Glue[END_REF][START_REF] Valentini | Histological Evaluation of Bio-Oss in a 2-Stage Sinus Floor Elevation and Implantation Procedure, A Human Case Report[END_REF]. In addition, the presence of proteins of bovine origin in certain xenograft materials, especially in Europe, is still being investigated in relation to the problem of the transmissible spongiform encephalopathy, a disease affecting cattle, but likely transmissible to humans [10].

A number of alloplastic materials have been developed to overcome many of the problems described here. These materials include calcium phosphates, calcium sulfates, and bioactive glasses [START_REF] Sailer | Bone Substitutes] Mund Kiefer Gesichtschir[END_REF][START_REF] Wetzel | Bone Apposition onto Oral Implants in the Sinus Area Filled with Different Grafting Materials, A Histological Study in Beagle Dogs[END_REF][START_REF] Low | An Evaluation of Bioactive Ceramic in the Treatment of Periodontal Osseous Defects[END_REF][START_REF] Zamet | Particulate Bioglass as a Grafting Material in the Treatment of Periodontal Intrabony Defects[END_REF][START_REF] Froum | Comparison of Bioactive Glass Synthetic Bone Graft Particles in the Treatment of Human Periodontal Defects, A Clinical Study[END_REF]. In general, these materials are said to be osteoconductive, and may or may not be resorbable. Different types of bioactive glass have been used clinically to regenerate bone loss as a result of periodontal disease for over 10 years. The bioactivity of these glasses was also verified for physical parameters, like surface morphology and granule size, and not only their chemical composition. As an example, we quote the work of Ducheyne [START_REF] Ducheyne | Effect of Bioactive Glass Particle Size on Osseous Regeneration[END_REF], who synthetized a glass called Biogran Õ that has the same chemical composition of PerioGlas Õ , and differs in the granule size range, 300-500 mm instead of 90-710 mm, and it is claimed to have a higher biocompatibility.

There has been a number of clinical studies that have demonstrated consistent results in treating intrabony defects [START_REF] Ong | Evaluation of a Bioactive Glass Alloplast in Treating Periodontal Intrabony Defects[END_REF]. One study showed that the clinical results of one type of bioactive glass were equivalent to DFDBA in treating two and three wall defects [START_REF] Lovelace | Clinical Evaluation of Bioactive Glass in the Treatment of Periodontal Defects in Humans[END_REF]. More recent studies have shown that when used to regenerate bone in fresh extraction sites or to augment the maxillary sinus, these materials fully resorb over a 6-to-13-month time period [START_REF] Froum | Histological Comparison of Healing Extraction Sockets Implanted with Bioactive Glass or Demineralized Freeze-dried Bone Allograft: A Pilot Study[END_REF][START_REF] Tadjoedin | Deproteinized Cancellous Bovine Bone (Bio-Oss) as Bone Substitute for Sinus Floor Elevation, A Retrospective, Histomorphometrical Study of Five Cases[END_REF]. The materials have been said to be osteostimulative [START_REF] Loty | Bioactive Glass Stimulates in vitro Osteoblast Differentiation and Creates a Favorable Template for Bone Tissue Formation[END_REF] in that they enhance the bone regeneration of the defect site.

The purpose of this study is to histologically and ultrastructurally evaluate three cases of bone augmentation procedures performed in the mandible, using bioactive glass with a subsequent dental implant placement and to report the resorbability of the bioactive glass and the bone regeneration in the implantation site.

MATERIALS AND METHODS

The cases included in this study and reported in Table 1 are all mandibular teeth that were diagnosed with advanced periodontal disease in need of extraction. All the patients were treated with the following medications: (1) Amoxicillin (1 g bid, for 1 day before tooth extraction and continued for 6 days), (2) Metasone retard (1 mg/day for 5 days) and (3) NSAID on demand bid. Probing depth was measured before tooth extraction in each case. The implant sites were prepared by drilling at a speed not exceeding 40 rpm and assuring torque at 32 N, without irrigation. This facilitated the harvesting of autogenous bone required to be mixed with bioactive glass (PerioGlas Õ , Novabone, USA) (composition: 45% (in weight per cent) SiO 2 , 24.5% CaO, 24.5% Na 2 O, 4% P 2 O 5 ; size: 90 -710 mm) and for a better control of implant alignment. Radiographs were obtained at various times including baseline, immediate post-operative and at 6, 12, 18 (data not shown) and 24 months. At each follow-up, routine hygiene was performed. With prior consent from the patients, biopsies were taken during the six-month follow-up visit.

Case 1 -The patient presented with a periodontal abscess of an endodontically involved molar with 3 furcation lesion having a probing depth of 7 mm, necessitating extraction (Figure 1(a)). The patient was prescribed prophylactic antibiotics for 1 week before extraction of 4.6

1 M 40 Good Yes <20 <20 2 M 60 Good No <20 <20 3 F 42 Good No >20 >20
GGHealth -Good general health; BOP -Bleeding on probing; FMPS -Full mouth plaque score.

Bone Augmentation with Bioactive Glass (lower right first molar). A full thickness flap was raised without vertical releasing incisions, without compromising the flap vascularization and the tooth was extracted, taking care to preserve the bone structure.

The socket was then thoroughly cleaned and the implant (Osseotite Õ Standard, 3i Implant Innovation, USA) placed in the interradicular bone. The implant length was chosen to ensure primary stability in the bone. The remaining socket of the extraction site, around the implant, was filled with a mixture of PerioGlas Õ and autogenous bone. The autogenous bone had been harvested from the drilling process during the preparation of the implant site. No additional sites were used to harvest bone (Figure 1(b)). A healing abutment was placed on the implant and the implant was covered for 6 months. At the time of placement of the abutment (6 months from surgery) a biopsy was performed distal to the abutment (Figure 1(c)). An additional small biopsy was taken from the soft tissue around the collar of the implant.

The healing abutment was replaced on the implant and the final restoration was made 2 weeks after the biopsy. The abutment was tightened at 32 N with a dynamometric tool controlled by endo-oral radiography (Trophy RGVui Digital Radiography System, Trophy Radiology, France) using Rinn technique and personal bite blocks to ensure consistent alignment. Case 2 -The patient presented with a high mobility of the cantilever bridge due to root fracture of 3.5 (lower left second premolar) (Figure 2(a)). The bridge was then removed and the tooth extracted and subsequently its socket was filled with a mixture of PerioGlas Õ and autogenous bone (Figure 2(b) and (c)) to maintain proper volume of bone for the esthetics of the final prosthesis. An implant could not be placed in the socket because of the lack of buccal bone, as an implant with dimensions of 4 mm diameter and 10 mm length would compromise the implant primary stability in the alveolar bone. A length more than 10 mm could penetrate the mandibular nerve canal, causing paresthesia. Full thickness flap with vertical mesial release incision was performed for a two-stage implant technique distally positioned to the graftfilled socket (Osseotite Õ Standard, 3i Implant Innovation, USA). The periosteum was released before suturing. At 6 months, a biopsy was taken from the filled socket and the implants were loaded. UCLA abutments (3i Implant Innovation, USA) were placed and tightened at 32 N using a dynamometric tool controlled by endo-oral radiography (Trophy RGVui Digital Radiography System, Trophy Radiology, France) using Rinn technique and personal bite blocks.

Case 3 -The patient presented with an advanced periodontal lesion as a result of poor oral hygiene and exhibited bleeding on probing (BOP) and full mouth plaque score (FMPS) >20%, and a probing depth of about 12 mm (Figure 3). In this case, 4.7 (lower right second molar) was extracted and the socket was filled with a mixture of PerioGlas Õ and the patient's blood. The graft site was then covered with a PLA membrane (Atrisorb Õ , Atrix Laboratories, USA) for better protection and the mesiovestibular periodontal pocket of 4.8 (lower right third molar) was also grafted as it presented a 7-mm probing depth. The periosteum was dissected to release the flap and to achieve a better primary healing of the wound.

A biopsy was taken after 6 months and two implants (Osseotite Õ Standard, 3i Implant Innovation, USA) were placed with a one-step technique keeping mounting devices in place for a better healing of the soft tissues. The implants were loaded after 6 months and intraoral radiographs (Trophy RGVui Digital Radiography System, Trophy Radiology, France) and orthopantomographs (Cranex Tome CEPH, Soredex, Finland) were taken during the two-year follow-up visit.

The biopsies were fixed in 4% paraformaldehyde and dehydrated in ethanol. After embedding in polymethylmethacrylate, the samples were sectioned with a diamond saw (Accutom, by Struers, Denmark) into a 200-mm thick and a 20-mm thick slice, respectively for scanning electron microscopy (SEM) (QUANTA-ESEM by FEI, The Netherlands) and for histological observation (Standard 25, Zeiss, Germany). The thin nondecalcified sections were stained with Stevenel's blue/Van Gieson's picrofuchsin or hematoxylin-eosin, suitable to show calcium deposition and bone morphology. The 200-mm thick sections were first microradiographed and then polished and prepared for SEM observations that were performed both in secondary emitted and backscattered mode (BSE). Elemental analyses and X-ray dot maps were carried out with an energy dispersive system (EDS, by EDAX, USA) to detect the topographic distribution of the elements in the PerioGlas Õ granules after implantation.

RESULTS

The histological and SEM analyses of the biopsy of Case 1 taken from the soft tissue around the implant collar show minimal inflammatory reaction; the cellular reaction around the particles is characterized by normal connective tissue with little infiltrate, demonstrating the compatibility of bioactive glass particles in contact with the soft tissues. The outer layer of the particles shows signs of infiltration and hematoxylin-eosin staining indicates the presence of a calcium-rich layer. The degradation also affects the particle core where cell colonization is observed (Figure 4). At low magnification under the SEM (Figure 5), the biopsy shows bone and granules that are not yet completely resorbed. They appear whiter since they are electronically denser than bone. The grey level contrast in the image clearly delineates older bone (lighter grey) from newly formed bone.

In the apical part of the biopsy, wide zones of mineralization are seen, probably due to autogenous bone pieces that were mixed with PerioGlas Õ during implantation and due to a more intimate contact with the bone walls. Trabecular bone was seen to have grown directly onto the surface of the glass particles away from the bone walls. Some granules appear to have an empty core indicating a resorption of the particles. In addition, new bone can be seen growing within the excavated pouch of the bioactive glass particle in the SEM image (Figure 6(a)). The appearance of the material adjacent to the newly formed bone is similar to precipitated calcium phosphate (Figure 6(b)).

After 6 months of implantation, the glass underwent a physicalchemical transformation and a significant part had already been transformed into calcium phosphate, as shown by the X-ray dot maps for silicon, calcium, and phosphorus (Figure 6(c)). The bioglass bioactivity mechanism was already described by many authors [START_REF] Gatti | Importance of the Microanalysis in Understanding the Mechanism of Transformation in the Active Glassy Materials[END_REF][START_REF] Gatti | Short Time Behaviour of Active Glass Granules in Bone Defects Repair[END_REF] and it could be divided into different steps: (a) ionic diffusive processes from the glass, (b) its transformation into a hydrated gel, (c) contradiffusion from the extracellular matrix toward the glass surface, and finally (d) precipitation of calcium phosphate in the glass.

Radiographic images of the implant site after 6 (Figure 1(c)) and 24 months (Figure 7) show good maintenance of the bone collar around implant.

Histological observation of the biopsy of Case 2 shows significant bone growth among the bioactive glass particles, which appear degraded: some are intimately connected to the newly formed bone while others are still surrounded by noninflammatory connective tissue. Figure 8 is an undecalcified histologic image (Van Gieson's picrofuchsin) of Bone Augmentation with Bioactive Glass bioactive glass particles inside the pocket. There was no indication of any inflammatory infiltrate.

Scanning electron micrographic investigations show new bone grown both on the particles surface (Figure 9, on the right side) and inside their core (Figure 9, on the left side) indicating an early stage of new bone formation.

Radiographic images show excellent maintenance of the bone collar around implants, even at two-year follow-up (Figure 10). Radiographic density of the regenerated bone appears as normal bone even during the early stages. At the final follow-up of these cases, the bone volume was maintained under loading of the implants when compared with the initial graft volume. In Case 2, where there was no implant placed, the volume was also maintained.

In Case 3, there was less bone formation, although the particle reactivity appeared to be comparable to the other cases. The biopsy in this case was taken from the crestal region and may not be representative of bone formation occurring more apically in the defect. The two-year post-operative clinical results (Figure 11) in this case are similar to the other cases and the implants have been successfully loaded and are stable. Thus, the early evidence of lower bone formation in the biopsy did not appear to have an adverse effect on the clinical result.

In two of the three cases, islands of bone formation were noted in the middle of the defect, far from the bone wall. Bone grows on the glass granule surface that works as a scaffold through the formation of a calcium phosphate-rich layer that induces the colonization of the bone cells.

DISCUSSION

From the results, it is very clear that the use of PerioGlas Õ has significantly influenced the prognosis in all the three cases described here. All the three cases were followed up at the end of two years in a completely loaded state and were all stable and healthy. PerioGlas Õ is one of the synthetic bone regenerative products belonging to the group of 'glasses and ceramics'. Though hydroxyapatite (HA) has been the most widely used alloplast, its prolonged resorption/ remodeling time and its brittleness have led to the development of other alloplasts with improved properties. PerioGlas Õ is made up of bioactive glass particles from 90 to 710 mm, and exhibits properties significantly superior to those of the other alloplasts. Like all synthetic grafts, PerioGlas Õ does not require a second surgery and there is no risk of disease transmission or immunologic response. The threedimensional scaffold formed in situ upon implantation is a critical determinant of the speed of remodeling and regeneration. PerioGlas Õ particulate imparts a greater surface area for the exposure of nutrients, vascularization, and bony ingrowth consequently leading to faster bone regeneration. Unlike most of the other alloplasts that are bioinert, PerioGlas Õ belongs to the class of bioactive regenerative materials. Not only does it act as an osteoconductive scaffold providing an appropriate environment in which bone cells and BMPs can adhere and proliferate, but also interacts with the surrounding tissues and imparts an osteostimulatory effect [START_REF] Gatti | Long Term Behaviour of Active Glasses in Sheep's Mandibular Bone[END_REF].

The ability of PerioGlas Õ to stimulate bone formation in the presence of osseous tissues but not in soft tissues makes it different from osteoinductive products. PerioGlas Õ is neither osteogenic nor osteoinductive, but a number of in vivo studies have demonstrated a more rapid bone formation with Bioglass particles. This has been attributed to the 'osteopromotion' and 'osteoproduction' principles, which are used interchangeably with 'osteostimulation.' Also, the viability and proliferation potential of osteoblasts (MG63) have been shown to be exemplified in the presence of bioglass particles [START_REF] Xynos | Ionic Products of Bioactive Glass Dissolution Increase Proliferation of Human Osteoblasts and Induce Insulin-like Growth Factor II mRNA Expression and Protein Synthesis[END_REF]. Because of its ionic release mechanism of action, PerioGlas Õ negates the acidic pH at the wound site, hence reducing the inflammation. In vitro tests have also shown that PerioGlas Õ can decrease the clotting time by at least 25%. More tests have proven a significant reduction (3 log) in the bacterial population. There have also been studies revealing an increased osteocalcin and alkaline phosphatase levels [START_REF] Price | Human Osteoblast-like Cells (MG63) Proliferate on a Bioactive Glass Surface[END_REF] at the recipient site in the presence of PerioGlas Õ , indicating that all these mechanisms ultimately help in providing a favorable site for osteoblast recruitment and osseous regeneration.

CONCLUSIONS

PerioGlas Õ shows very good handling properties during placement and does not require elaborate mixing techniques or equipment.

The surgical sites showed rapid healing of soft tissue and the material appeared to enhance hemostasis. The material appears to maintain graft volume and shows excellent biocompatibility.

These results show that bioactive glass particles are a viable synthetic graft material for use when grafting is necessary in conjunction with load bearing dental implants as it exhibits reliable and lasting results.

Figure 1 .

 1 Figure 1. (a) X-ray image of a grade 3 furcation involvement in the lower right first molar (see arrow); (b) implant site and socket filled with the bioglass granules after the implant procedure (see arrows); and (c) fixture and healing abutment 6 months after the implant. Arrows show the biopsy sites.

Figure 3 .

 3 Figure 3. Panoramic X-ray. Arrows indicate the 4.7 area before operation.

Figure 2 .

 2 Figure 2. (a) X-ray image of wide bone resorption due to root fracture (see arrow); (b) arrow shows the bone socket before bioactive glass implantation; and (c) bone socket filled with PerioGlas Õ granules plus autogenous bone (see arrows).

Figure 4 .

 4 Figure 4. Histologic hematoxylin-eosin stained section of the biopsy in the bone defect (10Â). Glass granules are still visible after 6 months from implantation (see arrows).

Figure 5 .

 5 Figure 5. Scanning electron micrograph of the total bone biopsy. Glass granules (whiter) are still visible in bone (see arrows).

Figure 6 .

 6 Figure 6. (a) SEM-BSE image of a degraded glass granule (whiter area) surrounded by new bone. Inside the particle there is a pouch where new bone is forming. The cracks cross the outer layer. The arrow indicates the interface of the glass granule-new grown bone. (b) EDS spectrum of the X-ray analyses in the outer layer showing mainly the presence of calcium and phosphorus. (c) X-ray dot maps for calcium (Ca), carbon (C), silicon (Si), and phosphorus (P) indicate the total glass transformation in a calcium phosphate.

Figure 7 .

 7 Figure 7. X-ray images of Case 1 implant site after 24-month follow-up.

Figure 8 .

 8 Figure 8. Histological view of a picrofuchsin-stained section. (10Â) No sign of adverse tissue reactions are visible.

Figure 9 .

 9 Figure 9. SEM-BSE image: arrows indicate degraded glass granules completely surrounded and invaded by new bone formation.

Figure 10 .

 10 Figure 10. X-ray image of Case 2 implant site after 24-month follow-up.

Figure 11 .

 11 Figure 11. Panoramic X-ray image of Case 3 after 24-month follow-up.

Table 1 .

 1 List of the cases.

	Case	Gender	Age	GG health	Smoker	BOP (%)	FMPS (%)

ACKNOWLEDGMENTS

The authors thank Mrs R. Salvatori for the technical help. The research was supported by University of Modena and Reggio Emilia.


Chiara Coppi

Chiara Paolinelli Devincenzi

Sergio Bortolini

Ugo Consolo

Roberta Tiozzo
email: tiozzo.roberta@unimore.it

A New Generation of Sterile and Radiopaque Impression Materials-an in vitro Cytotoxicity Study

Keywords: dental impression material, human gingival fibroblast, in vitro cytotoxicity tests, proliferation, viability

Impression materials are largely used to record the geometry of dental tissue. Hence, the assessment of their possible cytotoxicity is a necessary step in the evaluation of their biocompatibility. The present study is carried out to evaluate the cytotoxicity of a new elastomeric sterile and radiopaque impression material. Human gingival fibroblasts, cultured in vitro are exposed directly to Elite Implant in three different viscosities, heavy, medium, and light. At 3, 9, 24, 48, and 72 h, the cellular proliferation is evaluated. In parallel, human gingival fibroblasts are exposed indirectly by means of fluid extracts of Elite Implant. The cellular viability is evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, (MTT) assay (Sigma, St Louis, Mo). The gingival fibroblasts proliferation and viability are unaffected by the presence of Elite Implant. This new impression material may represent a safe medical device for clinical and surgical applications. In addition, this material is radiopaque and, thus, can be identified radiographically.

INTRODUCTION

A ll dental impression materials should accurately replicate intraoral and extraoral tissues in terms of accuracy, dimensional stability, elasticity, tear strength, rigidity, reproduction of detail, and biocompatibility [START_REF] Craig | Restorative Dental Materials[END_REF]. ISO 10993 is used to determine the safety of medical devices by defining various types of biocompatibility tests. Cellular toxicity testing, which is required for all types of medical devices, is covered in ISO 10993-5 [START_REF]Biological Compatibility of Medical Devices -Part 1[END_REF].

Four types of elastomers are extensively used: polysulfides, condensation silicones, polyethers, and vinyl polysiloxanes [START_REF] Brunton | Contemporary Dental Practice in the UK: Indirect Restorations and Fixed Prosthodontics[END_REF]. They are generally supplied in two paste forms, base and catalyst, and may be dispensed through an auto-mixing cartridge. Although polyethers present many advantages for clinical use, several disadvantages have been reported including allergic and toxic reactions, contact dermatitis, and gingivitis [START_REF] Nally | Hypersensitivity to a Dental Material: A Case Report[END_REF][START_REF] Craig | Characteristics and Clinical and Tissue Reactions of Impression Materials[END_REF][START_REF] Hensten-Pettersen | Guinea Pig Maximization Test with a Polyether Impression Material[END_REF][START_REF] Samaranayake | Carriage of Oral Flora on Irreversible Hydrocolloid and Elastomeric Impression Materials[END_REF][START_REF] Sydiskis | Cytotoxicity of Impression Materials[END_REF][START_REF] Tiozzo | Study of the Potential Cytotoxicity of Dental Impression Materials[END_REF][START_REF] Chen | Cytotoxicity of Dental Impression Materials[END_REF][START_REF] Ciapetti | Cytotoxicity Testing of Materials with Limited in vivo Exposure is Affected by the Duration of Cell-material Contact[END_REF]. Vinyl polysiloxanes are more commonly used [START_REF] Brunton | Contemporary Dental Practice in the UK: Indirect Restorations and Fixed Prosthodontics[END_REF]. Various studies have reported contradictory levels of toxicity of vinyl polysiloxanes, classified as toxic [START_REF] Samaranayake | Carriage of Oral Flora on Irreversible Hydrocolloid and Elastomeric Impression Materials[END_REF][START_REF] Sydiskis | Cytotoxicity of Impression Materials[END_REF][START_REF] Tiozzo | Study of the Potential Cytotoxicity of Dental Impression Materials[END_REF][START_REF] Chen | Cytotoxicity of Dental Impression Materials[END_REF], less toxic [START_REF] Sydiskis | Cytotoxicity of Impression Materials[END_REF], or non-toxic [START_REF] Tiozzo | Study of the Potential Cytotoxicity of Dental Impression Materials[END_REF][START_REF] Chen | Cytotoxicity of Dental Impression Materials[END_REF][START_REF] Ciapetti | Cytotoxicity Testing of Materials with Limited in vivo Exposure is Affected by the Duration of Cell-material Contact[END_REF]. The different grades of toxicity, evaluated in vitro, depend on the type of culture (primary cell or permanent cell lines), on the type of in vitro test (direct or indirect), and on the manufacturing processes of the materials.

The toxicity of dental impression materials is especially important when, during an intervention, a fragment becomes entrapped and remains within the gingival sulcus [START_REF] Marshak | Incidence of Impression Material Found in the Gingival Sulcus after Impression Procedure for Fixed Partial Dentures[END_REF][START_REF] Shen | Impression Materials[END_REF] under the suture, during impression making for implants or a surgical prosthesis. Fragments of impression materials have been found in soft tissues [START_REF] Schow | Trismus from Unusual Foreign Body in Pterygomandibular Space: Report of a Case[END_REF][START_REF] Prince | Impression Materials as Foreign Bodies[END_REF][START_REF] O'leary | Severe Periodontal Destruction Following Impression Procedures[END_REF][START_REF] Garey | An Unusual Foreign Body in the Buccal Vestibule: Report of a Case[END_REF][START_REF] Gullet | Residual Fragment of Rubber Base Material[END_REF][START_REF] Shiloah | Periodontal Hazards of Retained Impression Materials[END_REF][START_REF] Ree | An Unusual Swelling Following Endodontic and Prosthodontic Treatment of a Mandibular Molar due to a Foreign Body Reaction[END_REF], under the periosteum [START_REF] Prince | Impression Materials as Foreign Bodies[END_REF][START_REF] Olson | Foreign Body Removal: Report of a Case[END_REF][START_REF] Gettleman | Polysulfide Rubber Foreign Body: Report of a Case[END_REF][START_REF] Eliasson | Radiopacity of Impression Materials[END_REF], in the spongy bone [START_REF] Kent | Impression Material Foreign Body: Report of a Case[END_REF][START_REF] Eliasson | Rubber-base Impression Material as Foreign Body: Report of a Case[END_REF][START_REF] Glenwright | Bone Regeneration Following Damage by Polysulphide Impression Material[END_REF], and in the maxillary sinus [START_REF] Karanek | Foreign Body in the Antrum[END_REF][START_REF] Winstock | Impression Material Presenting in the Maxillary Antrum as a Foreign Body[END_REF][START_REF] Smith | Dental Impression Paste in the Maxillary Sinus[END_REF][START_REF] Lownie | Chronic Maxillary Sinusitis Resulting from Displacement of Impression Material into the Maxillary Antrum: A Case Report[END_REF]. This can also occur in implant dentistry, particularly during second stage implant surgery or during single stage surgery. The retention of these fragments can induce a severe inflammatory reaction [START_REF] Prince | Impression Materials as Foreign Bodies[END_REF][START_REF] O'leary | Severe Periodontal Destruction Following Impression Procedures[END_REF][START_REF] Garey | An Unusual Foreign Body in the Buccal Vestibule: Report of a Case[END_REF][START_REF] Ree | An Unusual Swelling Following Endodontic and Prosthodontic Treatment of a Mandibular Molar due to a Foreign Body Reaction[END_REF][START_REF] Gettleman | Polysulfide Rubber Foreign Body: Report of a Case[END_REF][START_REF] Glenwright | Bone Regeneration Following Damage by Polysulphide Impression Material[END_REF], which can result in implant failure.

An in vivo study recently reported postoperative problems following root canal treatment [START_REF] Ree | An Unusual Swelling Following Endodontic and Prosthodontic Treatment of a Mandibular Molar due to a Foreign Body Reaction[END_REF]. One week after filling, the patient presented with a painful swelling associated with the treated tooth. Following clinical examination, a thin piece of residual polyether impression material left in the tissues was identified as the possible cause of the swelling. This material was transparent, and not detected by radiographic examination. A clinical report highlights the importance of the radiopacity of any impression material for assisting in location and retrieval of accidentally embedded materials [START_REF] Eliasson | Radiopacity of Impression Materials[END_REF].

The purpose of this study was to investigate the cytotoxicity of a new vinyl polysiloxane, which is both sterile and radiopaque.

MATERIALS AND METHODS

Impression Materials

The heavy, medium, and light forms of Elite Implant (Zhermack, Badia Polesine, Rovigo, Italy), which are both sterile and radiopaque, were studied.

Cell Culture

Human gingival fibroblasts were chosen because they represent a good simulation of the in vivo condition particularly in cases of impression material retention [START_REF] Kasten | A Model Culture System with Human Gingival Fibroblasts for Evaluating the Cytotoxicity of Dental Materials[END_REF] in the gum.

Human gingival fibroblasts were isolated from biopsies of a healthy subject, after providing informed consent, who was undergoing gingivectomy of the molar region. Immediately after removal, the biopsies were placed in collection medium consisting of Hank's balanced salt solution (HBSS) (Gibco, Grand Island, New York), containing 250 UI/mL penicillin and 0.25 mg/mL streptomycin, 0.05 mg/mL gentamycin, and 0.0025 mg/mL amphotheracin B (all from Gibco). The fibroblasts were grown as previously described [START_REF] Tiozzo | Study of the Potential Cytotoxicity of Dental Impression Materials[END_REF][START_REF] Kasten | A Model Culture System with Human Gingival Fibroblasts for Evaluating the Cytotoxicity of Dental Materials[END_REF][START_REF] Lang | The Use of Cultures of Human Osteoblast like Cells as an In Vitro Test System for Dental Materials[END_REF][START_REF] Mosmann | Rapid Colorimatric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays[END_REF]. The cells were cultured in Dulbecco's modification of Eagle's medium (DMEM) (Gibco), supplemented with 10% fetal bovine serum (FCS) (Gibco), 50 UI/mL penicillin, 50 mg/mL streptomycin, 2 mM L-glutamine (Gibco), and 1mM Na piruvate (Sigma, St Louis, Mo) at 37 C in humidified atmosphere, 95% air, and 5% CO 2 . The culture reached confluence in 7 d and was then subcultured until the experiments were initiated. In the study, the cultures were used to the sixth passage.

Measurements of Cytotoxicity

The direct and indirect tests of cytotoxicity were carried out following pre-defined methodology [START_REF] Sydiskis | Cytotoxicity of Impression Materials[END_REF][START_REF] Mosmann | Rapid Colorimatric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays[END_REF][START_REF] Twentyman | A Study of Some Variables in a Tetrazolium Dye (MTT) Based Assay for Cell Growth and Chemiosensitivity[END_REF] with some modifications [START_REF] Tiozzo | Study of the Potential Cytotoxicity of Dental Impression Materials[END_REF].

Direct Cytotoxicity Test

Human gingival fibroblasts, 20 Â 10 4 , were plated, in duplicate, in 60 mm petri dishes (Falcon, BD Bioscience, Milano, Italy) in a total volume of 5 mL of culture medium with 10% FCS and antibiotics, both in the presence and the absence of impression materials and their components. The polymerized impression materials (1 cm 2 ) were placed in the center of the petri dishes under sterile conditions. The dishes were incubated at 37 C under a humidified atmosphere of 95% air and 5% CO 2 . Cell proliferation was evaluated after 3, 9, 24, 48, and 72 h. At each time period the medium was removed, cells in monolayer were trypsinized (Na-EDTA trypsin) (Gibco) for 7-10 min, and counted in a hemocytometer (Neubauer). Data were analyzed statistically using Student's test and statistical significance was set up, p<0.05. Reported values are the mean of four replicates AE standard deviation.

Indirect Cytotoxicity Test

The indirect test was performed by incubating impression materials (1 cm 2 ) in petri dishes, in 5 mL of culture medium without FCS for 24 h at 37 C under sterile conditions. At the end of the incubation period, the soluble extracts of these impression materials were collected in tubes and enriched with 10% FCS (Gibco). Human gingival fibroblasts were plated at 20 Â 10 3 per well in 24-well plates (Falcon; BD Bioscience) in 2 mL of culture medium. When the cell cultures were sub-confluent, the medium was removed; the cell monolayer was washed with PBS and exposed to diluted (50%) or undiluted (100%) soluble extracts at 12 and 24 h. Each concentration was tested in quadruplicate wells together with appropriate controls. At the end of the treatment, cell viability was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, St Louis, Mo) [START_REF] Tiozzo | Study of the Potential Cytotoxicity of Dental Impression Materials[END_REF][START_REF] Twentyman | A Study of Some Variables in a Tetrazolium Dye (MTT) Based Assay for Cell Growth and Chemiosensitivity[END_REF]. The MTT assay is an indirect marker for cytotoxicity. It is based on the ability of cells to reduce the slightly yellow soluble MTT to a deep purple formazan product, insoluble in aqueous solution. The reaction is catalyzed by mitochondrial succynil dehydrogenase.

After 24 h of cell culture in the presence of undiluted or diluted extracts of impression materials, the medium was removed and 2 mL of growth medium with 200 mL of MTT (5 mg/mL in PBS) were added to the cultures. Cells were incubated, in the dark, at 37 C in a humidified atmosphere (95% air and 5% CO 2 ) for 3 h. The growth medium was removed, 2 mL of dimethyl sulfoxide (DMSO; Sigma) were added to each well to dissolve the purple formazan. The absorbance was measured in a spectrophotometer at a wavelength of 540 nm. This parameter is an indicator of the functional integrity of the mitochondria, and hence of cellular viability. Reported values are the mean of four replicates AE standard deviation and are expressed as percentages of the control values.

Morphological Analysis

Human gingival fibroblasts were observed through an inverted microscope in phase contrast mode at Â50 magnification (Nikon Diaphot-TMD) and photographed after 3, 9, 24, 48, and 72 h of culture both in the presence and the absence of impression materials and their components.

RESULTS

Direct Effect of Elite Implant Polymerized and its Components -Base and Catalyst Pastes -On the Proliferation of Human Gingival Fibroblasts

The direct effect of polymerized Elite Implant heavy viscosity was evaluated at 3, 9, 24, 48, and 72 h in culture. The impression materials did not inhibit cell proliferation at 48 h. At 72 h, Elite Implant heavy viscosity induced a negligible inhibitory effect, 8 AE 1% lower than that found in control specimens (Figure 1). Elite Implant medium viscosity also produced a negligible inhibitory effect, 13 AE 4% lower than that found in the control cells at 48 h, and at 72 h, the Elite Implant medium catalyst produced a clear inhibitory effect, %19 AE 5% (Figure 2). Similar effects on cell proliferation were obtained by Elite Implant light viscosity and its components (Figure 3).

Indirect Effect of Elite Implant on the Viability of Human Gingival Fibroblasts Cultured in vitro

The diluted (50%) and undiluted (100%) extracts obtained after incubation for 24 h of the heavy, medium, and light Elite Implant materials did not induce any alteration in cellular viability (Figure 4).

Morphologic Analysis of Human Gingival Fibroblasts Cultured in the Presence of Elite Implant Heavy Viscosity Polymerized and its Components -Base and Catalyst Pastes

At 48 h, human gingival fibroblasts, grown without impression material, were uniformly distributed and elongated in the petri dishes, showing typical fibroblast morphology (Figure 5A). In the presence of polymerized Elite Implant heavy viscosity (Figure 5B) and its components, base and catalyst pastes (Figure 5C andD), the culture of fibroblasts, did not show any morphological alteration. The growth and morphology of cells were similar to that of the control culture with some mitosis observed. At 72 h, the gingival fibroblasts were homogeneously distributed on the substrate and produced a complete monolayer. The cells increased in number and appeared with a narrow and elongated morphology (Figure 6A). The morphology of gingival fibroblasts grown in the presence of Elite Implant Heavy viscosity polymerized and its components is similar to that of the control cells (Figure 6B-D).

DISCUSSION

The aim of the present study was to evaluate the cytotoxicity of Elite Implant (Zhermack), a new elastomeric, sterile, and radiopaque impression material.

In vitro cytotoxicity tests were developed to simulate and predict biological reactions to the materials placed into or on human tissues. Considering this aim, particular care should be taken to select cell types and experimental conditions. Continuous cell lines are easy to maintain; they grow quickly and show good reproducibility. They are simple replicating systems without the specific metabolic potential that the target cells have in vivo. Moreover, continuous lines are sensitive and useful to test and classify the toxic effect of different materials [START_REF] Lang | The Use of Cultures of Human Osteoblast like Cells as an In Vitro Test System for Dental Materials[END_REF]. In the present study, the cytotoxicity of this new dental material was assessed using human gingival fibroblasts cultured in vitro. These cells are characterized by a high degree of differentiation even if they are less homogeneous and sensitive than permanent cell lines; they are more comparable in their reaction pattern to oral mucosa. Human gingival fibroblasts retain specialized features and can represent a good simulation of the in vivo condition particularly in the case of impression material retention [START_REF] Kasten | A Model Culture System with Human Gingival Fibroblasts for Evaluating the Cytotoxicity of Dental Materials[END_REF]. Elite Implant was found not to affect cellular proliferation and viability. This result confirms other reports [START_REF] Tiozzo | Study of the Potential Cytotoxicity of Dental Impression Materials[END_REF][START_REF] Chen | Cytotoxicity of Dental Impression Materials[END_REF][START_REF] Marshak | Incidence of Impression Material Found in the Gingival Sulcus after Impression Procedure for Fixed Partial Dentures[END_REF] and also indicates that the manufacturing process and the components, added to the material in order to make it sterile and radiopaque, do not make it cytotoxic to IIa. Because of its sterility, it has an elective use in operating theaters, specific indications include regenerative bone surgery, intraoperative impressions, immediately loaded implant-supported prostheses, and when making impressions of sub-gingival implant platforms. Finally, the bright color (blue or yellow) of Elite Implant may help the dentist to visualize fragments left intraorally.

Figure 1 .

 1 Figure 1. Direct effect of Elite Implant heavy viscosity polymerized and its components, base and catalyst pastes, on in vitro proliferation of human gingival fibroblasts. Each point represents the mean of four measurements AE SD.

Figure 2 .

 2 Figure 2. Direct effect of Elite Implant medium viscosity polymerized and its components, base and catalyst pastes, on in vitro proliferation of human gingival fibroblasts. Each point represents the mean of four measurements AE SD.

3 Figure 3 .

 33 Figure 3. Direct effect of Elite Implant light viscosity polymerized and its components, base and catalyst pastes, on in vitro proliferation of human gingival fibroblasts. Each point represents the mean of four measurements AE SD.

Figure 4 .

 4 Figure 4. Indirect effect of diluted (50%) and undiluted (100%) extracts of Elite Implant heavy, medium, and light viscosity obtained after 24 h of incubation on cell viability of human gingival fibroblasts. Data are expressed as a percentage of optical density (AESD) compared with control cells (% of control).

Figure 5 .

 5 Figure 5. Morphological analysis of human gingival fibroblasts cultured for 48 h in the absence of impression material (A); in the presence of polymerized Elite Implant heavy viscosity (B); in the presence of base paste (C); and in the presence of catalyst paste (D). Cell cultures were observed by inverted microscopy in phase contrast mode at Â50 magnification.

Figure 6 .

 6 Figure 6. Morphological analysis of human gingival fibroblasts cultured for 72 h in the absence of impression material (A); in the presence of polymerized Elite Implant heavy viscosity (B); in the presence of base paste (C); and in the presence of catalyst paste (D). Cell cultures were observed by inverted microscopy in phase contrast mode at Â50 magnification.

ACKNOWLEDGMENT

This study has been conducted with the help of grants from Italian MURST-MUIR and Zhermack, Badia Polesine, Rovigo, Italy.


Zorica Ajdukovic ´

Nenad Ignjatovic

Dragan Petrovic

Dragan Uskokovic ´2

Substitution of Osteoporotic Alveolar Bone by Biphasic Calcium Phosphate/Poly-DLlactide-co-glycolide Biomaterials

Keywords: osteoporosis, alveolar bone, reconstruction, biphasic calcium phosphate/poly-DL-lactide-co-glycolide (BCP/PLGA), biocomposites

Lost bone tissue due to osteoporosis makes dentistry very difficult. The aim of this study is to reconstruct the bone tissue with composite biomaterials and to estimate the optical density and alveolar ridge height of the mandible. Research is conducted on 30 postmenopausal women aged from 46 to 62 years, with diagnosed osteoporosis and defects in alveolar bones caused by extraction of paradontopathic teeth, enucleation of cysts and periapical changes, extraction of impacted teeth, or by trauma. Biphasic calcium phosphate/poly-DL-lactide-co-glycolide (BCP/PLGA) composite is implanted into the defects of alveolar bones. Six weeks after implantation of BCP/PLGA, the alveolar bone density in the region of premolars on the experimental side of the jaw is found to be lower than that on the untreated, control, side of the jaw. On the contrary, 24 weeks after implantation, it is significantly higher compared with the density of the control side. A significant increase in optical density of alveolar bones in the region of premolars on the experimental side compared with the control one is noticed. These results indicate a high level of osteoregeneration and osteoblast activity. Synthetic BCP/PLGA composite belongs to the group of biomaterials, which facilitate formation of new bones

INTRODUCTION

O steoporosis is a systemic bone disease, characterized by reduction in bone volume per volume unit [START_REF] Won Wowern | Osteoporosis: A Risk Factor in Periodontal Disease[END_REF][START_REF] Klemetti | A Review of Residual Ridge Resorption and Bone Density[END_REF]. Bone provides support and stability to all types of dental implants and suprastructures. Therefore, problems with deossification caused by osteoporosis have been seriously addressed in stomatology [START_REF] Ravn | Association between Pharmacokinetics of Oral Ibandronate and Clinical Response in Bone Mass and Bone Turnover in Women with Postmenopausal Osteoporosis[END_REF][START_REF] Antundal | The Effect of Alendronate on Resorption of the Alveolar Bone Following Tooth Extraction[END_REF][START_REF] Jahangari | Current Perspectives in Residual Ridge Remodelling and its Clinical Implications: A Review[END_REF], because any loss of bone tissue has as a consequence inadequate dentistry [START_REF] Jahangari | Current Perspectives in Residual Ridge Remodelling and its Clinical Implications: A Review[END_REF][START_REF] Polla ¨hne | Osteoporose -Bildgebende Diagnostik[END_REF][START_REF] Becker | A Prospective Clinical Trial of Endosseous Screw-shaped Implants Placed at the Time of Tooth Extraction without Augmentation[END_REF].

Numerous procedures for the reconstruction of alveolar bones have been reported. So far, repairs to alveolar bone defects have been made in monkeys, dogs, and rats [START_REF] Hu ¨rzeler | Guide Bone Regeneration around Dental Implants in the Atrophic Alveolar Ridge using a Bioresorbable Barrier -An Experimental Study in the Monkey[END_REF][START_REF] Caulier | The Relationship of Some Histologic Parameters, Radiographic Evaluations, and Periotest Measurements of Oral Implants: An Experimental Animal Study[END_REF][START_REF] Hahn | Preservation of the Alveolar Ridge with Hydroxyapatite-collagen Implants in Rats[END_REF]. In clinical studies, correction of alveolar deformities, height and width of alveolar ridges, has been made by destructive osteogenesis [START_REF] Zaffe | Morphofunctional and Clinical Study on Mandibular Alveolar Distraction Osteogenesis[END_REF]. Some authors have reported on porous, tricalcium phosphates (-TCP) used as filler for large alveolar defects of the mandible after enucleation of residual cysts, to achieve regular alveolar ridge height so that dental implants can be easily inserted [START_REF] Ajdukovic | Repair of Bone Tissue Affected by Osteoporosis Hydroxyapatite-poly-l-lactide (HAp/PLLA) with and without Blood Plasma[END_REF].

In order to solve the problem of bone tissue loss caused by advanced resorption of alveolar bone due to systemic osteoporosis, we have used a biphasic calcium phosphate/poly-DL-lactide-co-glycolide (BCP/DLPLG) composite biomaterial [START_REF] Ignjatovic | New Biocomposite [Biphasic Calcium Phosphate/Poly-DL-lactide-co-glycolide/Biostimulative Agent] Filler for Reconstruction of Bone Tissue Changed by Osteoporosis[END_REF]. The most important characteristic of biphasic calcium phosphate is its chemical similarity to the mineralized bone phase, while bioresorbable polymers based on lactide and glycolide exhibit a high degree of biocompatibility and nontoxicity [START_REF] Nagano | Bone Bonding Ability of an Apatite-coated Polymer Produced using a Biomimetic Method: A Mechanical and Histological Study in vivo[END_REF][START_REF] Tanahashi | Apatite Coating on Organic Polymers by Biomimetic Process[END_REF][START_REF] Fournier | Biocompatibility of Implantable Synthetic Polymeric Drug Carriers: Focus on Brain Biocompatibility[END_REF]. Synthetic BCP/PLGA composite belongs to the group of biomaterials, which induce easier formation of new bones, and due to its osteoconductive potential it can successfully replace the bone tissue [START_REF] Elgendy | Osteoblastlike Cell (MC3T3-E1) Proliferation on Bioerodible Polymers: An Approach towards the Development of a Bone-Bioerodible Polymer Composite Material[END_REF].

In previous work [START_REF] Ignjatovic | New Biocomposite [Biphasic Calcium Phosphate/Poly-DL-lactide-co-glycolide/Biostimulative Agent] Filler for Reconstruction of Bone Tissue Changed by Osteoporosis[END_REF], the artificial defects in mandibles of rats damaged by osteoporosis were filled with BCP/PLGA biocomposite in order to reconstruct them. As BCP/PLGA in these investigations proved to be an extremely good osteoconductive biomaterial in osteoreconstructive processes, it was then used in clinical research.

The aim of this study is to investigate the possibility of reconstructing the bone tissue using calcium phosphate/poly-DL-lactide-co-glycolide composite biomaterial and to repair alveolar bones of patients affected by osteoporosis. The results of these applications were analyzed during the postoperative period by measuring relative optical densities of bone tissues and the heights of alveolar ridges. The novelty, compared to previous studies, is the reconstruction of alveolar bones of patients with diagnosed osteoporosis. The reparative ability of BCP/PLGA to restore the deformed alveolar ridge and the structure of prosthetic supporting tissue, which plays the primary role in the formation of prosthetic suprastructure, i.e., insertion of dentures that are to recover lost esthetic, phonetic, and masticatory functions, was examined.

MATERIALS AND METHOD

The used BCP/PLGA composite was synthesized according to the procedure given earlier [START_REF] Ignjatovic | New Biocomposite [Biphasic Calcium Phosphate/Poly-DL-lactide-co-glycolide/Biostimulative Agent] Filler for Reconstruction of Bone Tissue Changed by Osteoporosis[END_REF][START_REF] Ignjatovic | Synthesis, Characterization and Application of Composite Biomaterials Biphasic Calcium Phosphate/Poly-dl-lactide-co-glycolide as Filler and Blocks for Reparation Hard Bone Tissue[END_REF]. Calcium phosphate gel was obtained by precipitation of calcium nitrate and ammonium phosphate in an alkaline medium. It was dried at room temperature and then calcined at 1100 C for 6 h. Poly-DL-lactide-co-glycolide (PLGA) (50 : 50) (Sigma Chemical Co., St. Louis, USA) was used as a polymer component. Into completely dissolved polymer, calcium phosphate granules (80 mass percent) were added. After stirring (30 rev/min), methanol was added to the solution. Then, the solvent was removed and the particles were dried at room temperature for 24 h. Composite biomaterial particles 0.153-0.2 mm in size were sieved and sterilized by -rays (25 kGy) before use.

Upper and lower jaw tissue of 30 postmenopausal women aged 46-62 years was studied. After having been diagnosed with periapical lesions, impacted teeth, and cysts in alveolar bones, the patients were prepared for surgery (mucoperiosteal flap was lifted, pathologic changes eliminated, and the lost bone tissue replaced with BCP/PLGA composite). Then, the diagnosed parodontopathic teeth in the premolar or molar region were extracted and the extraction wounds filled with BCP/PLGA composite. One side of the jaw (the experimental one), A, was filled with BCP/PLGA composite, while the other, contralateral one, served as the control side, B.

Radiological analysis included radiography of jaws by orthopanthomographical method. Density and height of alveoli were analyzed in the regions of premolars and molars, i.e., regions from which pathologic tissue was removed. The heights of these regions, from the lower edge of the jaw to the upper edge of the defect, were measured by a Manser (Inox) ruler (nonius). The relative optical density was measured in an area 14 mm in diameter around the defect. Each orthopanthomogram had a fixed, transparent, thermo-stable celluloid foil with a printed net of squares (2 Â 2 mm 2 ). On each foil, regions of first premolars and molars were marked. A light beam from a densitometer DT II 05 (London, Great Britain) was passed through each segment of the orthopanthomograph. Numerical values of the relative optical densities were calculated and statistically analyzed. After consolidation and healing of wounds, fixed or mobile dentures, depending on the state of the prosthetic supporting tissue and the number and condition of the remaining teeth, were inserted. The patients were observed during a period of 6-24 weeks.

The statistical significance of differences was analyzed by Student's t-test.

RESULTS AND DISCUSSION

As already mentioned, patients' bones weakened by osteoporosis were reconstructed with BCP/PLGA composite (Figure 1). Osteoporosis was diagnosed by a densitometer [START_REF] Tezal | The Relationship between Bone Mineral Density and Periodontitis in Postmenopausal Women[END_REF] and also by blood biochemical analyses. The obtained results for calcium of 1.8 mmol/L, phosphorus of 0.69 mmol/L, and alkaline phosphatase of 58.1 IU/L confirmed the disease.

A composite production procedure via a solvent-non-solvent system provides covering of BCP particles with DLPLG polymer. Figure 1 shows SEM images of BCP/PLGA composite particles. As evident, BCP granules with an average particle diameter of 0.15-0.20 mm are coated with the polymer [START_REF] Ignjatovic | New Biocomposite [Biphasic Calcium Phosphate/Poly-DL-lactide-co-glycolide/Biostimulative Agent] Filler for Reconstruction of Bone Tissue Changed by Osteoporosis[END_REF][START_REF] Ignjatovic | Synthesis, Characterization and Application of Composite Biomaterials Biphasic Calcium Phosphate/Poly-dl-lactide-co-glycolide as Filler and Blocks for Reparation Hard Bone Tissue[END_REF]. The principal idea of application is that, after implantation of the composite biomaterial into the bone defect, the polymer component (PLGA) that reinforces BCP bioresorbs thus BCP/PLGA was implanted in the premolar or molar region of the experimental side of the jaw (A), while the control, contralateral, side (B) (also premolar or molar region) was healthy and untreated. Analyses (measurement of alveolar ridge height and relative optical density) were made around the center of defects 14 mm in diameter; the same was carried out on the control side. Differentiation can also be made between the existing and newly-formed bone on the basis of the degree of their illumination. Newly formed bone, after implantation of BCP/PLGA is surrounded by a halo.

The healthy, control, side of the jaw is denoted in all figures by an arrow and letter B. The experimental side is denoted by A.

After impacted canine tooth and odontoma (shown in Figure 2(a)) had been diagnosed, the patient was prepared for the preprosthetic surgical A cyst in the molar region of the alveolar bone of the mandible can be seen in Figure 3(a). Six weeks after enucleation of the cyst and implantation of BCP/PLGA, the formation of new bone tissue as well as a slight increase in optical density and alveolar ridge height, which is statistically still considerably lower than those of the control side of the jaw, can also be seen (Figure 3(b)). Twenty-four weeks after surgery, the formation of new bone tissue, initiated by intensive osteoregenerative processes, very similar to the tissue of the control side of mandible, as well as an increase in relative optical bone density and alveolar ridge height, is evident as shown in Figure 3(c). After healing of the wound and consolidation of the prosthetic supporting tissue, a mobile denture The values summed up in Table 1 reveal a statistically significant decrease in optical density of alveolar bone in the region of premolars on the experimental side of the jaw (A) contrary to the density of the control side (B) (P < 0.01) six weeks after implantation of BCP/PLGA. These relative optical density values indicate the beginning of osteogenesis in alveolar bones weakened by osteoporosis, but they are still much lower than those of the control, untreated sides of jaws, due to insufficient time for bone tissue regeneration. In the 24th week (according to Figure 2(b)), a significant increase in optical density of the alveolar bone in the premolar region of the experimental side (A) contrary to the control side (B) (P < 0.05) can be seen. This confirms prevalence of osteoregenerative processes, increased activity of osteoblasts [START_REF] John | Surface Reactivity of Calcium Phosphate based Ceramics in a Cell Culture System[END_REF], decrease in atrophy and resorption, and improvement in prosthetic supporting tissue after implantation of the BCP/PLGA composite and adequately inserted dentures. Atrophy and resorption of the alveolar bone, which can be caused by systemic osteoporosis, other reasons, or diseases, present a serious problem in prosthetic rehabilitation. To solve this problem, porous hydroxyapatite granules together with polyglactin 910 Õ have been used by some authors, who, applying the vestibuloplastic method, obtained satisfactory results [START_REF] Sugar | Augmentation of the Atrophic Maxillary Alveolar Ridge with Hydroxylapatite Granules in Vicryl(Polyglactin) Knitted Tube and Simultaneous Open Vestibuloplasty[END_REF].

Six weeks after implantation of BCP/PLGA, the analyzed alveolar ridge heights in the premolar regions on the experimental sides (A) taken from orthopanthomographs are lower than those of the control sides (B), but these differences are statistically insignificant (Table 1). Disturbances in bone metabolism in the premolar region are of lower intensity than those in the molar region, because this part of the jaw is subjected to less loads than the molar region, so that the rehabilitation is much faster for the same period. A slight increase in bone tissue resorption (as a self-protection reaction of the organism) was also Substitution of Osteoporotic Alveolar Bone by BCP/PLGA observed during the first 6 weeks after implantation of the biocomposite, but soon after that osteogenesis prevailed. Twenty-four weeks after implantation and treatment of patients with adequate dentures, a statistically significant increase in heights of alveolar ridges in the premolar regions on the experimental sides of jaws (A) compared with the control ones (B) (P < 0.01) was observed. Also, a faster consolidation and healing of wounds after extraction were noticed. These results indicate the positive effects of combined treatments: the implantation of BCP/PLGA composite and the use of adequate dentures.

During the six weeks after implantation of the biocomposite, a trabecular network was formed with collagen as the main structural protein, but six weeks is too short a period for deposition of a significant amount of inorganic salts and formation of good quality bone. In the beginning, BCP/PLGA composite causes a slow deposition of hydroxyapatite crystals, which follow collagen fibers orientation, while faster and better quality bone formation rich in inorganic components was observed 24 weeks after implantation of BCP/PLGA. Therefore, the relative optical density and alveolar ridge height, six weeks after implantation, are statistically significantly lower than those of the control side of the jaw, but 24 weeks after implantation of BCP/PLGA they are statistically much higher compared with the density and height of the control side, which is in agreement with the previous results.

Table 2 summarizes the values of densities and heights of alveolar ridges in the molar regions. A statistically significant decrease in optical densities of bones in the molar regions on the experimental sides of jaws (A), contrary to those of the control sides (B) (P < 0.01), can be seen 6 weeks after BCP/PLGA implantation. These values indicate already started rehabilitation and osteogenesis of tissues damaged by osteoporosis, but they are still much lower than the values obtained for the control sides (Figure 3(b)). Disturbed bone metabolism was first registered in the regions around lower molars, where stresses during mastication are the most pronounced, the reason why 6 weeks is an insufficient period for the regeneration and increase in density of such a loaded bone tissue. In the 24th week, a significant increase in relative optical densities of bones in the molar regions of the experimental (A) contrary to the control sides (B) (P < 0.05) is observed.

The results indicate a significant increase in intensity of regenerative processes in the alveolar bone that lead to the reconstruction of the alveolar ridge, which is in accordance with the results obtained by some authors who studied deproteinized beef bone, xenograft, and osteosynthesis, but taking into account trabecular bone volume, jaw region, and the sex of patients [START_REF] Artzi | The Application of Deproteinized Bovine Bone Mineral for Ridge Preservation Prior to Implantation. Clinical and Histological Observations in a Case Report[END_REF][START_REF] Wiesen | Preservation of the Alveolar Ridge at Implant Sides[END_REF][START_REF] Malek | The Bioconvertible Osteosynthesis System -Result of a Prospective Clinical Study on Stability of Bimaxillary Surgery[END_REF][START_REF] Ulm | Characteristic Features of Trabecular Bone in Edentulous Maxillae[END_REF]. Our results show an improved structure as well as quality of the alveolar bone affected by osteoporosis (as can be seen in Figure 3(c)), because after implantation of BCP/PLGA composite, incremental advances in new-formed vascular tissue, increased activity, and adherence of osteoblasts capable of providing much faster reparative processes in the bone affected by osteoporosis took place. All these led to improved prosthetic supporting tissue and successfully inserted dentures (Figure 3(d)).

The effects of mentioned treatments (implantation of the BCP/PLGA composite and insertion of adequate dentures) induced an increase in osteoblast activity, improvement in the structure and quality of the alveolar bone affected by osteoporosis, as well as local increase in bone density and alveolar ridge height in the premolar or molar region, where the composite had been implanted. By providing patients with adequate dentures, resorption in the regions subjected to increased stress was avoided, while increased activity of osteoblasts and rebuilding of alveolar bones prevailed. Similar results were obtained by other authors who reconstructed alveolar ridges using other implant materials [START_REF] Jahangari | Current Perspectives in Residual Ridge Remodelling and its Clinical Implications: A Review[END_REF][START_REF] Sugar | Augmentation of the Atrophic Maxillary Alveolar Ridge with Hydroxylapatite Granules in Vicryl(Polyglactin) Knitted Tube and Simultaneous Open Vestibuloplasty[END_REF][START_REF] Anselme | Osteoblast Adhesion on Biomaterials[END_REF]. The use of resorbable BCP/PLGA biomaterials facilitated the growth of newly formed vascular tissue, fibroblasts, and increased activity and adhesion of osteoblasts [START_REF] Legeros | Properties of Osteoconductive Biomaterials: Calcium Phosphates[END_REF].

Alveolar bone is the most unstable paradoncium tissue and belongs to the group of the most dynamic human tissues, constantly rebuilding or being resorbed, which is a result of its adaptation to the homeostatic and mechanical needs of the organism [START_REF] Won Wowern | Osteoporosis: A Risk Factor in Periodontal Disease[END_REF][START_REF] Ravn | Association between Pharmacokinetics of Oral Ibandronate and Clinical Response in Bone Mass and Bone Turnover in Women with Postmenopausal Osteoporosis[END_REF][START_REF] Antundal | The Effect of Alendronate on Resorption of the Alveolar Bone Following Tooth Extraction[END_REF]. Lower values of blood biochemical parameters, especially alkaline phosphatase, indicate lower osteoblast activity, retardation of anabolic processes, and appearance of osteoporosis. When depositing bone matrix, osteoblasts secrete small quantities of alkaline phosphatase, which causes a decrease in local concentration of inorganic phosphate, inhibition of collagen fiber activity, and decreased deposition of calcium salts [START_REF] Riis | Biochemical Markers and Bone Turnover in Diagnosis and Assessment of Therapy[END_REF]. Loss of the residual alveolar ridge usually connected with systemic or local osteoporosis, paradontal diseases, surgical procedures, tooth extraction, or congenital defects can lead to permanent deformities. During cicatrization of the defects, soft tissue usually collapse into them. The consequences of such a cicatrization are inadequate dentures, both functionally and aesthetically; in some cases it is even impossible to insert them. Therefore, correction of the alveolar ridge during tooth extraction or preprosthetic surgical preparations, before inserting dentures, provides optimal results in prosthetic rehabilitation. Application of bioresorbable BCP/DLPLG composite biomaterial facilitates generation of new bone tissue and its penetration through the very material, which leads to successful prosthetic treatment.

CONCLUSION

Based on investigations carried out, it can be concluded that BCP/ PLGA, 6 weeks after its implantation in the premolar and molar region of the experimental side of the jaw, causes a decrease in optical density and height of alveolar ridges of patients. In the 24th week, however, a significantly higher optical density and greater alveolar ridge height in the premolar and molar region on the side where the composite was implanted, compared with the control one, was found. The obtained results also indicate a more intensive osteoregenerative process and high level of osteoblast activity on the side where the composite was implanted. With increasing osteogenesis, in alveolar bones weakened by osteoporosis, the density of bone increases, so that the quality of the prosthetic supporting tissue after implantation of the composite is improved and can bear the dentures inserted.

Figure 1 .

 1 Figure 1. SEM images of BCP/PLGA composite biomaterial particles.

Figure 2 .

 2 Figure 2. Rehabilitation of defects in the alveolar bone of maxilla: (a) the presence of impacted canine and odontoma in the region of first premolars (A -experimental side, B -control side); (b) formation of new bone tissue after extraction of impacted tooth and odontoma, and implantation of BCP/PLGA into the generated defect; (c) consolidation of prosthetic supporting tissue; and (d) inserted fixed, upper denture.

Figure 3 .

 3 Figure 3. Rehabilitation of defects in the alveolar bone of the mandible: (a) a cyst in the alveolar bone of the mandible in the molar region (A -experimental side, B -control side); (b) formation of new bone tissue after cyst enucleation and implantation of BCP/ PLGA into the generated defect; (c) consolidation of prosthetic supporting tissue of the mandible; and (d) insertion of a mobile, partial, lower denture.

Table 1 .

 1 Values of relative optical densities and alveolar ridge heights in the region of premolars.

	Experimental side of jaw (A)

*All values are mean AE SD.

Table 2 .

 2 Values of relative optical density and alveolar ridge heights in the region of molars.

	Experimental side of jaw (A)

*All values are mean AE SD.

Substitution of Osteoporotic Alveolar Bone by BCP/PLGA


Alan M Smith

Richard M Shelton

Yvonne Perrie

Jonathan J Harris
email: j.j.harris@bham.ac.uk

An Initial Evaluation of Gellan Gum as a Material for Tissue Engineering Applications

Keywords: gellan, hydrogel, bone, cell viability, three-dimensional

Alpha-modified minimum essential medium (MEM) has been found to cross-link a 1% gellan gum solution, resulting in the formation of a selfsupporting hydrogel in 1:1 and 5:1 ratios of polysaccharide: MEM. Rheological data from temperature sweeps confirm that in addition to orders of magnitude differences in G 0 between 1% gellan and 1% gellan with MEM, there is also a 20 C increase in the temperature at which the onset of gelation takes place when MEM is present. Frequency sweeps confirm the formation of a true gel; mechanical spectra for mixtures of gellan and MEM clearly demonstrate G 0 to be independent of frequency. It is possible to immobilize cells within a threedimensional (3D) gellan matrix that remain viable for up to 21 days in culture by adding a suspension of rat bone marrow cells (rBMC) in MEM to 1% gellan solution. This extremely simple approach to cell immobilization within 3D constructs, made possible by the fact that gellan solutions cross-link in the presence of millimolar concentrations of cations, poses a very low risk to a cell population immobilized within a gellan matrix and thus indicates the potential of gellan for use as a tissue engineering scaffold.

(TE) substrates in the past with several studies examining materials, such as alginate, chitosan, and gelatin as cell scaffolds for both two-dimensional (2D) and three-dimensional (3D) cell culture [START_REF] Pan | Chitosan-based Scaffolds for Hepatocyte Culture[END_REF][START_REF] Park | BMP-2 Induces the Expression of Chondrocyte-specific Genes in Bovine Synovium-derived Progenitor Cells Cultured in Three-dimensional Alginate Hydrogel[END_REF][START_REF] Barralet | Comparison of Bone Marrow Cell Growth on 2D and 3D Alginate Hydrogels[END_REF][START_REF] Zhang | A Sandwich Tubular Scaffold Derived from Chitosan for Blood Vessel Tissue Engineering[END_REF][START_REF] Li | Demineralized Bone Matrix Gelatin as Scaffold for Osteochondral Tissue Engineering[END_REF]. An advantage in using hydrogel-forming biopolymers for the creation of 3D culture matrices is that the high water content of such materials facilitates the diffusion of nutrient and signaling molecules both to and from the cells seeded within and upon a hydrogel matrix.

Three-dimensional (3D) cell culture as a precursor to tissue creation is necessary because cells do not spontaneously assemble into complex 3D structures after seeding on a flat surface; some form of external support is required. The ability to maintain cells in a viable state, allowing nutrient/waste exchange is a key requirement of any scaffold material employed in this supporting role. The biological relevance of 3D culture as opposed to conventional 2D culture was highlighted by Hishikawa et al. [START_REF] Hishikawa | Gene Expression Profile of Human Mesenchymal Stem Cells During Osteogenesis in Three-dimensional Thermoreversible Gelation Polymer[END_REF] who demonstrated that the spatial relationship between cells seeded within a 3D matrix had a direct bearing upon gene expression.

Many polysaccharide solutions may be converted into gels by exposure to cations. Alginate is cross-linked by Ca 2þ ions to form a gel with physical properties for a given ratio of guluronic acid: mannuronic acid being determined by the initial solution strength and the concentration of cations available for cross-linking. A variety of studies have demonstrated that incorporation of cells into a liquid solution of alginate, which is then dripped into a salt solution, can result in the creation of alginate beads with immobilized cells that remain viable within the 3D construct [START_REF] Mehlhorn | Mesenchymal Stem Cells Maintain TGFbeta-mediated Chondrogenic Phenotype in Alginate Bead Culture[END_REF]. However, while such approaches have demonstrated that cells may be maintained in a viable state, there exists the risk that viability may be compromised through exposure of cells to relatively high ionic concentrations during and after cross-linking to obtain gels of suitable strength (as might be necessary where constructs of greater complexity than a bead are required). Other routes to cell immobilization have explored the possibility of thermal gelation phenomena in alternative polysaccharides and again, although the extent of cell viability has been encouraging it would intuitively be advantageous to avoid exposure of cells to fluctuations in temperature in order to maximize the chance of survival.

Gellan gum is an extracellular polysaccharide, secreted by the bacteria Sphingomonas paucimobilis (formerly Sphingomonas elodea) during aerobic fermentation [START_REF] Fialho | Structures and Properties of Gellan Polymers Produced by Sphingomonas paucimobilis ATCC 31461 from Lactose Compared with Those Produced from Glucose and from Cheese Whey[END_REF] that has been extensively employed within the food industry as a stabilizer and thickening agent and is commonly referred to in this application (within the European Union) as E418. At elevated temperatures (e.g., %80 C for a 1% solution) the polymer exists in solution as a disordered coil which is converted to a double helix structure [START_REF] Chandrasekaran | The Crystal Structure of Gellan[END_REF][START_REF] O'neill | Structure of the Acidic Extracellular Gelling Polysaccharide Produced by Pseudomonas elodea[END_REF] when cooled. This helical structure is not a true gel network although in this ordered state gellan can exhibit 'weak gel' characteristics. Formation of a true gel network is reliant upon aggregation of helical sequences, which on the basis of current evidence [START_REF] Morris | Conformational and Rheological Transitions of Welan, Rhamsan and Acylated Gellan[END_REF] can occur via different mechanisms dependent on the cation used in cross-linking; divalent cations, such as Ca 2þ and Mg 2þ form direct bridges by site binding between pairs of double helices, monovalent ions, such as Na þ and K þ suppress repulsion by binding to the surface of the helices balancing the negative charge of the carboxyl groups. The concentrations required to induce gelation are therefore different and depend on the gellan concentration, but typically, concentrations of %5 mM Ca 2þ and Mg 2þ , %100 mM for Na þ and K þ would be required for cross-linking.

In addition to various forms of gellan used within the food industry, gellan gum is commercially available in a form that is guaranteed to be free of endotoxin allowing for its use in medical formulations. The main medical application to date has been for controlled drug delivery both as an encapsulating agent [START_REF] Kedzierewicz | Effect of the Formulation on the in vitro Release of Propranolol from Gellan Beads[END_REF] and as an active ingredient in an ophthalmic formulation that gels on exposure to the tear film, thus ensuring the medications remain suitably located in the eye [START_REF] Sultana | Ion-activated, Gelrite (R)-based in situ Ophthalmic Gels of Pefloxacin Mesylate: Comparison with Conventional Eye Drops[END_REF]. Gellan has been used in plant tissue culture [START_REF] Klimaszewska | Plantlet Development from Immature Zygotic Embryos of Hybrid Larch through Somatic Embryogenesis[END_REF][START_REF] Nakano | Adventitious Shoot Regeneration and Micropropagation of Hybrid Tuberous begonia (Begonia  tuberhybrida Voss)[END_REF], bacterial culture [START_REF] Rule | Gellan Gum as a Substitute for Agar in Leptospiral Media[END_REF] and has been evaluated as a component of a composite material for mammalian TE [START_REF] Ciardelli | Blends of Poly-(epsilon-caprolactone) and Polysaccharides in Tissue Engineering Applications[END_REF][START_REF] Cascone | Bioartificial Polymeric Materials Based on Polysaccharides[END_REF]. However, there have been no reports of gellan being used exclusively for the immobilization and culture of mammalian cells. The present study investigated whether exposure of gellan gum to MEM would result in gelation, with a view to possible application in TE where cell immobilization within a synthetic extracellular matrix was required.

MATERIALS AND METHODS

Preparation of Polysaccharide Solutions

Endotoxin-free low-acyl gellan gum (Gelrite,CP Kelco Inc, USA, Lot#: 4I1690A) was dissolved in deionized water (dH 2 O) at 80 C with continuous stirring to a solution strength of 1% wt/wt.

Cell Isolation and Incorporation into Gellan Constructs

Rat bone marrow cells (rBMC) were isolated and cultured according to the method described by Maniatopoulos et al. [START_REF] Maniatopoulos | Bone Formation in vitro by Stromal Cells Obtained from Bone Marrow of Young Adult Rats[END_REF]. Briefly, femora from mature albino Wistar rats (around 120 g in weight) were dissected out removing as much as adherent soft tissue as possible. The epiphyses were removed and the femora were repeatedly flushed with minimum essential medium (MEM) (Sigma, UK) containing 10% fetal bovine serum (FBS), 2.5% HEPES, 10% penicillin/streptomycin (P/S), and 1% amphotericin. Cells were then incubated in a 75 mL flask using supplemented MEM medium containing 10% FBS, 2.5% HEPES, 1% P/S in a humidified atmosphere of 95% air, and 5% CO 2 at 37 C. After 7 days of primary culture, cells were dissociated with 0.25% trypsin-0.02% EDTA (Sigma, UK). The cells were centrifuged at 1500 rpm (400 g) for 5 min before resuspension at a density of 2.3 Â 10 5 cells/mL in MEM and mixed with 1% gellan gum followed by light agitation to promote mixing of the two solutions.

Assessment of Rheological Properties

Rheological analysis was carried out in the linear viscoelastic region using a 1 /55 mm cone and plate geometry mounted on a Malvern Gemini Rheometer (Malvern Instruments, UK) fitted with Peltier plate thermal control. Temperature sweeps were conducted at a rate of 1 /min from 60 to 10 C, with a fixed oscillation frequency of 1 rad/s and a strain of 1%, while frequency sweeps to assess the mechanical properties of the material at varying strain rates were conducted at an isotherm of 37 C, in auto-stress mode with a fixed strain of 1%.

Assessment of Cell Viability

Cell viability was evaluated using a Live/Dead Viability/Cytotoxicity Kit [L-3224] (Molecular Probes, Invitrogen, USA) following culturing rBMC immobilized in 1 mL gellan matrix for periods of up to 21 days. This stain reflects the uptake of calcein AM and conversion to fluorescent calcein (green) by intracellular esterases in viable cells and the binding of ethidium bromide (red) to the DNA of cells with damaged plasma membranes.

RESULTS

Construct Formation

Where MEM and gellan were mixed in equal quantities prior to extrusion through an aperture of 1.7 mm a mechanically unstable composition resulted which rapidly dissociated into isolated lumps of gel in an excess of MEM. The proportions were varied before identifying 0.2 mL cell suspension in 1 mL 1% gellan solution (1:5 volume ratio) as an appropriate mixing regime to produce self-supporting constructs. Mixing of the gellan solution with cell suspension was carried out thereafter within a 5 mL disposable syringe at 37 C, the mixture being subject to vibratory mixing before being extruded into an excess of MEM to form self-supporting cylindrical structures as illustrated in Figure 1.

Rheological Characteristics

Measurements of storage (G 0 ) and loss (G 00 ) modulus in samples of 1% gellan are provided in Figure 2 which depicts the changes in rheological Evaluation of Gellan Gum for Tissue Engineering Applications behavior on cooling of 1% gellan, 1% gellan with an equal volume of MEM, and a control specimen comprising a 1:1 mixture of 1% gellan with distilled water (dH 2 O). Mechanical spectra derived from frequency sweeps are shown in Figure 3, illustrating behavior consistent with a solution (1% gellan with dH 2 O), a weak gel (1% gellan), and a true gel in which G 0 is independent of frequency (1% gellan with MEM).

Cell Viability

Constructs arising from 1 : 1 mixtures of gellan and cells suspended in MEM exhibited nearly 100% viability at 3, 6, 10, and 21 days (10 day culture illustrated in Figure 4). Although occasionally cells fluorescing red were visible amongst the predominance of green fluorescence, they were isolated and were only identified following lengthy and detailed examination of the constructs and did not correspond to any particular location within the constructs. Validation of the live/dead assay in terms Rheology: temperature sweeps. The elastic (storage) and viscous (loss) moduli (G 0 and G 00 , respectively) of a 1% gellan solution as compared with a specimen containing equal volumes of 1% gellan and MEM and another containing equal volumes of 1% gellan and distilled water (dH 2 O). Of significance is that where 1% gellan was supplemented with an additional liquid the same volume of liquid was used in both cases; however, in the case of MEM the ionic concentration was sufficient to outweigh the dilution that took place.

of ensuring that the gellan construct did not inhibit the diffusion of the ethidium bromide component to entrapped cells was carried out by immersing a portion of a construct in 70% ethanol for 15 min to bring about cell death. The assay was then performed and on inspection was found to have resulted in exclusively red staining being observed under fluorescence microscopy (Figure 4) indicating a lack of integrity of the plasma membrane highlighted by binding of the ethidium bromide homodimer with the nucleus of affected cells, in turn supporting the veracity of the assay when used upon 1% gellan gum constructs containing cells. Self-supporting cylindrical constructs arising from 1 : 5 mixtures of MEM: gellan demonstrated a more mixed response when assayed, showing an increased tendency for cell aggregation in comparison to the 1:1 mixtures, as might be expected given the cell density of the suspension employed was identical in both cases (2.3 Â 10 5 cells/mL). Some additional 1 : 5 constructs were seeded with substantially increased cell density of 7.9 Â 10 6 cells/mL, equating to a total of 1.58 Â 10 6 cells per 1.2 mL gellan construct. In this case a more aggressive vibratory mixing regime was employed wherein an air bubble was incorporated into the mixture to act as a mixing ball, in an attempt to improve the distribution of cells within the construct. Although live cells were visible throughout the 1 : 5 constructs, the density of cells within the gellan matrix was much reduced (initial suspension density 2.3 Â 10 5 cells/mL). In addition, evidence of cell death was observed, typically in areas where multiple cells were clustered together in a relatively small volume. However, where the higher seeding density (7.9 Â 10 6 cells/mL) was employed in conjunction with vigorous mixing this problem appeared to be eliminated; such constructs exhibited even cell distribution and a similar predominance of green fluorescence as had been observed for the original 1:1 mixtures.

DISCUSSION

Construct Formation

The mechanical instability of constructs comprising 1 : 1 gellan: MEM is likely to have arisen from disruption of helical junction zones within the gel matrix during extrusion due to the shear forces that would have been induced within the polysaccharide matrix. In contrast, reduction of the availability of cations resulting from a reduced volume of MEM added to gellan (0.2 mL in 1 mL, respectively) allowed formation of gelled cylinders on extrusion into excess MEM. A possible reason for the difference was due to the gellan being in the ordered form but not fully aggregated in the 1 : 5 mixture, thus a reduced proportion of junction zones were disrupted on shearing. At a 1:1 ratio full aggregation had occurred, which could not be recovered post-shear in spite of an excess of MEM being present; it is inferred that cations had occupied all available binding sites on the gellan molecule. Where reduced quantities of MEM were added followed by mixing, a heterogeneous mixture of gelled regions (aggregated gellan helices) within a solution of ordered but not aggregated gellan helices was formed. It is proposed that subsequent extrusion into excess MEM resulted in aggregation of polymer helices through ionotropic crosslinking induced as the fresh supply of cations began to diffuse into the partially formed construct, entrapping the previously aggregated helices in a gelled matrix. Indirect evidence existed to support this suggestion as a degree of cell aggregation is noted in 1 : 5 mixtures that was absent in the 1:1 mixtures, where the distribution of cells was considerably more uniform throughout the construct. This was probably related to the fact that cells were introduced as a suspension in MEM and so became entrapped within a cation-rich region of gellan that was subsequently shattered upon mixing and dispersed through the ungelled remainder, as discussed above. However, with the use of a more aggressive mixing regime involving an incorporated air bubble these problems were eliminated; cell distribution improved to a point where no differences could be seen between the 1:1 and 1:5 constructs.

Cross-linking of gellan with MEM occurred as a result of the availability of cations within MEM, which as supplied by Sigma Aldrich UK were present in the following concentration: NaH 2 PO 4 (1 mM), NaCl (116 mM), KCl (5.4 mM), MgSO 4 (0.75 mM), and CaCl 2 (1.8 mM). It is presently unclear precisely which cations were responsible for gelation; however, it has previously been demonstrated that all of those listed as constituents of MEM are capable of causing gelation of 1% solutions of gellan [START_REF] Grasdalen | Gelation of Gellan Gum[END_REF], with stronger gels resulting from gel formation in the presence of Ca 2þ and Mg 2þ as opposed to the monovalent cations, such as Na þ and K þ .

Rheological Characteristics

Temperature sweeps (Figure 2) illustrated shifts in the temperature at which the onset of gelation occurred within each sample. In the case of 1% gellan and 1% gellan diluted with dH 2 O the several orders of magnitude increases in G 0 were consistent with disordered-to-ordered transitions within the polysaccharide chains, while aggregation of ordered helices occurred where cations were present from the addition of MEM, pushing the gelation temperature above 50 C. With the addition of dH 2 O the increased dilution of the gellan solution (effectively creating a 0.5% gellan solution) resulted in a reduction in the temperature at which increases in G 0 were observed, those increases being consistent with the adoption of helical conformations by polysaccharide chains. Where MEM was added to gellan an identical volume to that of dH 2 O in the control was employed, hence in effect the system was diluted to an equal extent in the case of MEM as originally for dH 2 O. However, the supply of cations from MEM was sufficient to induce aggregation of helical domains (cross-linking) which outweighed the dilution that took place in terms of the response of the material to applied strain. The mechanical spectra illustrated in Figure 3 supported this finding; gellan with MEM in a 1 1 ratio had characteristics of a true gel, namely that G 0 was clearly dominant over G 00 and was independent of the frequency of oscillation. In contrast, the mechanical spectra of 1% gellan suggested the formation of a weak gel that arose through the adoption of helical domains within gellan chains, but no subsequent aggregation due to a lack of available cations. The frequency sweep of gellan with dH 2 O exhibited no gel characteristics whatsoever, instead this mechanical spectrum was typical of a solution of entangled polymer chains. Noticeable for both 1% gellan and 1% gellan þ dH 2 O were some significant fluctuations in measured G 0 and G 00 values where frequency <5 Hz, commensurate with spurious interpretations of inertia during rheological testing, probably arising from the combination of relatively high frequencies of oscillation and relatively low viscosities of the specimens under test.

Cell Viability

Three-dimensional cultures based on 1 : 1 mixtures assayed for intracellular esterase activity or damaged plasma membranes revealed the presence of live cells at all depths within the construct, for all culture times. In contrast, some cell death was observed where cell clustering had occurred in the 1 : 5 gelled mixtures, which was eliminated on mixing as has been previously discussed. The difference in the spatial distribution of cells within the two types of construct before the more aggressive mixing regime was adopted is likely to be behind the observed differences in viability, as it is well known that cell aggregates are prone to apoptosis.

In general terms the survival of cells within gellan constructs is highly encouraging with respect to the future prospects for the use of gellan gum for TE purposes. However, in isolation live/dead assays provide a rather limited assessment of the impact on cells of immobilization in a gellan matrix and certainly do not address the question of how a mammalian host would respond to the implantation of cell-loaded gellan constructs, which is a long-term aim. However, at the present stage the authors are primarily interested in ways in which culture of cells in three dimensions can be achieved for the purposes of in vitro study, and to this end gellan exhibits some promise.

GENERAL DISCUSSION

The potential for gellan in TE goes far beyond what can be achieved in terms of cell viability in a 1% solution cross-linked with MEM. Variations in the structure of the tetrasaccharide molecule (i.e., high or low acyl forms, which may be mixed in a range of ratios) provide for extensive tailoring of rheological characteristics; the respective formation of everything from soft elastic gels to firm brittle gels. Consequently, there is scope for creating culture matrices of varying gel texture that will allow exploration of the response of cells to the texture of a 3D environment in which they are immobilized along the lines of recent studies [START_REF] Cukierman | Taking Cell-matrix Adhesions to the Third Dimension[END_REF][START_REF] Panorchan | Microrheology and ROCK Signaling of Human Endothelial Cells Embedded in a 3D Matrix[END_REF][START_REF] Winters | Three-dimensional Culture Regulates Raf-1 Expression to Modulate Fibronectin Matrix Assembly[END_REF][START_REF] Zaman | Migration of Tumor Cells in 3D Matrices is Governed by Matrix Stiffness Along with Cellmatrix Adhesion and Proteolysis[END_REF], just as the response of cells to textured surfaces has been extensively explored with 2D cultures [START_REF] Berry | The Influence of Microscale Topography on Fibroblast Attachment and Motility[END_REF][26][START_REF] Hamilton | Microfabricated Discontinuous-edge Surface Topographies Influence Osteoblast Adhesion, migration, Cytoskeletal Organization, and Proliferation and Enhance Matrix and Mineral Deposition in vitro[END_REF][START_REF] Liliensiek | The Scale of Substratum Topographic Features Modulates Proliferation of Corneal Epithelial Cells and Corneal Fibroblasts[END_REF]. Control over rheological properties suggests that gellan could be used both in injectable and indirect (in vitro culture followed by implantation) approaches to TE, with injectable routes taking advantage of the gelation behavior of gellan under physiological conditions. Inspection of immobilized cells to considerable depths using conventional microscopy is aided by the excellent optical clarity of gellan gels and the capacity to process a cell loaded construct using polymerase chain-reaction (PCR) to identify markers of gene expression could be greatly aided by the fact that gellan gum appears not to inhibit PCR [START_REF] Rath | Gellan Gum as a Suitable Gelling Agent in Microbiological Media for PCR Applications[END_REF] unlike other polysaccharides, such as alginate and agar [START_REF] Wadowsky | Inhibition of PCR-Based Assay for Bordetella-Pertussis by Using Calcium Alginate Fiber and Aluminum Shaft Components of a Nasopharyngeal Swab[END_REF][START_REF] Gibb | Inhibition of PCR by Agar from Bacteriological Transport Media[END_REF].

CONCLUSIONS

Gellan gum represents a potentially useful addition to the range of polysaccharide hydrogels that may be employed as tissue engineering (TE) substrates, particularly given that media-driven gelation or gelation under physiological conditions represents a very low risk route to a cellular payload. There is considerable scope for further work to more fully explore the potential of gellan gum for TE and to exploit the wider research possibilities offered by this extremely simple route to cell entrapment in a 3D hydrogel matrix.

  Figures 1 and 4 appear in color online: http://jba.sagepub.com INTRODUCTION B iopolymers have received attention as tissue engineering

Figure 1 .

 1 Figure 1. Construct creation. Photograph illustrating the self-supporting nature of gellan cylinders created through extrusion of 1% gellan gum solution from a 5 mL syringe (aperture 1.7 mm) into MEM. The cylinders produced were manually positioned in this example, demonstrating that cylinders were not only self-supporting, but were sufficiently robust to permit handling with forceps.

Figure 2 .

 2 Figure2. Rheology: temperature sweeps. The elastic (storage) and viscous (loss) moduli (G 0 and G 00 , respectively) of a 1% gellan solution as compared with a specimen containing equal volumes of 1% gellan and MEM and another containing equal volumes of 1% gellan and distilled water (dH 2 O). Of significance is that where 1% gellan was supplemented with an additional liquid the same volume of liquid was used in both cases; however, in the case of MEM the ionic concentration was sufficient to outweigh the dilution that took place.

Figure 4 .Figure 3 .

 43 Figure 4. Cell viability. Photomicrographs obtained using fluorescence microscopy illustrating (left) rBMC immobilized within a gellan gum matrix, compared with the experimental control (right) in which rBMC immobilized within a gellan gum matrix were fixed with ethanol prior to performing the assay. Exclusively green fluorescence (left) indicated intracellular esterase activity while red fluorescence (right) indicated binding of the ethidium bromide homodimer with the nucleus of cells with disrupted plasma membranes. This result confirmed that a gellan gum matrix does not inhibit the diffusion of the ethidium bromide components of the assay to cells within the matrix.

ACKNOWLEDGMENTS

The authors are grateful to CP Kelco (USA) for the kindly donated sample of gellan gum and also to the Engineering and Physical Sciences Research Council, whose Engineering Instrument Pool loaned the Malvern Gemini rheometer that allowed our mechanical characterization to proceed.