A biodegradable gelatin-based nanostructured sponge with space maintenance to enhance long-term osteogenesis in maxillary sinus augmentation

Abstract

The search for bone substitutes that are biodegradable, ensure space maintenance, and have osteogenic predictability, is ongoing in the field of sinus augmentation. We thus compared the bone regeneration potential of nanostructured sponges (NS-Sponge) with that of collagen-stabilized inorganic bovine bones (BO-Collagen), gelatin sponges (Gelatin), and blood clots (Cont) in sinus augmentation of rabbits. NS-Sponge was prepared by thermally induced phase separation with porogen leaching techniques. All the materials were non-hemolytic and cytocompatible. The porous and nanofibrous NS-Sponge showed better dimensional stability to support cell growth and osteogenic differentiation. In vivo, the sinus membrane collapsed in Cont and Gelatin, while BO-Collagen and NS-Sponge maintained the elevated height as assessed by come-beam computed tomography. Limited bone regeneration was observed in Cont and Gelatin. In the entire implanted area, histological analysis revealed a higher percentage of new bone area at 4 weeks of BO-Collagen treatment; however, a significantly greater increase in new bone area was observed after 12 weeks of NS-Sponge treatment. The 12-week remnant NS-Sponge material was significantly lower than the 4-week remnant material. Overall, NS-Sponge may be highly recommended for sinus augmentation, as it exhibits numerous advantages, including excellent operability, clear imaging characteristics, space maintenance, biodegradability, and superior osteogenic potential.

Keywords

Sinus augmentation bone substitute nanostructured gelatin biodegradable Bio-oss collagen

Introduction

Elevation of the maxillary sinus floor is the most widespread approach to increase alveolar bone height in edentulous sites of the posterior maxilla and enable endosseous dental implant placement.^1,2 Maxillary sinus floor elevation can be achieved by either creating a lateral window or performing alveolar osteotomy.³ After raising the sinus membrane, the bone substitute is implanted inter-positionally between the sinus membrane and the bony wall. Autogenous bone is usually considered as the gold standard among bone substitutes.⁴ However, intra-oral surgical sites only provide limited amount of autogenous bone ⁵ and the harvesting procedure is associated with postoperative complications and discomfort.^6,7 In addition, sinus grafting with autogenous bone presents unpredictable re-pneumatization, generating inadequate bone volume for further implant placement.^8,9 Therefore, bone substitutes with a slower resorption rate are proposed to be advantageous in maintaining space and rebuilding bone tissue in the sinus cavity.

BO-Collagen is a commercial bone substitute comprising 10% porcine type-1 collagen stabilized inorganic deproteinized bovine bone mineral (Bio-oss collagen®), available in block form. The low-resorbing inorganic deproteinized bovine bone mineral (BO; Bio-Oss®) is demonstrated to have high osteoconductivity,¹⁰ while the type-1 collagen matrix promotes cell proliferation, migration, differentiation, and mineralization.^11,12 The low-resorbing bovine bone material provides superior three-dimensional (3 D) maintenance,¹³ which is a desirable characteristic compared with autogenous bone in maxillary sinus augmentation. Conflicting reports have discussed the long-term performance of BO; some question the prognosis of dental implants put in bone with graft material residues,¹⁴ whereas others stated that grafted particles do not interfere dental implant osseointegration.^15,16 Although the final destination and resorption time of bone substitutes are not well understood, clinicians have reached a consensus that they tend to insert implants in natural bone tissue. However, this animal-derived material may be associated with pathogen transmission and can burden patients with high medical costs.¹⁷ These limitations prompted clinicians to explore alternate bone graft materials that are cost efficient, biosafe, and allow a suitable resorption rate that matches the rate of bone rebuilding.

Gelatin is a form of denatured collagen protein that has a similar chemical composition as collagen and can be considered as its macromolecule alternative.¹⁸ Moreover, the denaturing process eliminates potential pathogens in gelatin and upgrades its biosafety.¹⁹ Gelatin improves the adhesion, differentiation, and proliferation of cells by the arginine-glycine-aspartic (RGD) sequence,²⁰ is sensitive to matrix metalloproteinases (MMPs); and the products formed during its enzymatic degradation are biocompatible.²¹ Gelatin sponge is a 3 D scaffold commonly used as a cost-efficient alternative for bone grafts in dental clinics. It is sponge-like and fits in specific craniomaxillofacial cavities, such as the dental socket and maxillary sinus.^22,23 Despite its excellent proposed characteristics, the weak mechanical strength and rapid degradation of gelatin limit its clinical application.²⁴

A 3 D gelatin-based nanostructured sponge (NS-Sponge) is fabricated using thermally induced phase separation and porogen leaching technique (TIPS&P).²⁵ This nanostructured gelatin sponge has superior characteristics than traditional gelatin sponge, such as a nanofibrous topography, high porosity, and interconnected pores. The nanofibrous topography and 3 D architecture of the gelatin scaffold is demonstrated to substantially enhance osteogenic differentiation of embryonic stem cells in vitro.²⁶ In addition, cross-links stabilize the nanofibrous structure, improve mechanical strength, and slow degradation rate in vivo. The existing knowledge of NS-Sponge suggests its prospect use in sinus augmentation.

To the best of our knowledge, this study is the first to use the gelatin-based NS-Sponge in maxillary sinus augmentation. It aimed to investigate the validity of gelatin-based NS-Sponge in guiding new bone formation in the sinus microenvironment of rabbits. Imaging features, trabecular bone parameters, and histological results were analyzed to evaluate the quantity and quality of newly formed bone following a sinus floor elevation procedure. Knowledge on this type of scaffold-guided bone formation in the maxillary sinus may present future alternatives for bone substitutes in clinical dental surgery.

Materials and methods

Preparation of NS-Sponge

The NS-Sponge was prepared through thermally induced phase separation with porogen leaching techniques.²⁵ Paraffin spheres (0.4 g) sized 250 − 420 µm were preheated in Teflon molds at 37 °C for 40 minutes. Gelatin (2.0 g) was dissolved in water (10 mL) and ethanol (10 mL) compound at 45 °C. Subsequently, the prepared gelatin solution was poured onto paraffin spheres and immediately transferred to a temperature environment of -76°C for 4 h to achieve phase separation. After solvent exchange and freeze-drying, the gelatin/paraffin composite was tailored to the required size (5.0 mm diameter and 5 mm thickness). The paraffin spheres were leached out at 37 °C in a hexane solution, which was replaced six times every 12 h. The scaffolds were crosslinked with 1-ethyl-3–(3-dimethylaminopropyl) carbodiimide HCl (EDC) and N-hydroxy-succinimide (NHS) at 4 °C for 24 h. An acetone/water (90/10, v/v) solvent mixture was used to maintain the nanostructure and inhibit swelling of the gelatin scaffolds. After being washed three times with distilled water at 37 °C and frozen at -20°C for 12 h, the scaffolds were freeze-dried for 3 days. The scaffolds were then sterilized with 75% ethanol for 2 h and washed with phosphate buffered saline (PBS, Gibco) thrice (30 min each) to elute the remaining ethanol.

Characterization of gelatin sponge, Bio-oss collagen®, and NS-Sponge

A gelatin sponge (Absorbable Gelatin Sponge, Jinling, Nanjing, China), collagen-stabilized inorganic bovine bone (Bio-oss collagen®, Geistlich AG, Switzerland), and an NS-Sponge were viewed under scanning electron microscopy (SEM; TM-1000, Hitachi, Japan) to evaluate their surface morphology. The samples were sputter-coated with gold prior to the analysis. The mechanical properties of the three materials were analyzed with wet samples to evaluate their flexibility by monitoring resilience after removal of external compression.

Hemolysis assay

A hemolysis assay was used to evaluate the blood compatibility of the NS-Sponge, BO-Collagen, and Gelatin. A blood sample was collected from the marginal ear vein of New Zealand rabbits. The anti-coagulated blood was centrifuged and washed in sterile isotonic saline. Red blood cells (RBCs) were then resuspended in a 2% (v/v) solution using sterile isotonic saline. The materials were soaked in diluted RBCs. Sterile distilled water and sterile isotonic saline served as positive and negative controls, respectively. After incubation at 37 °C for 2 h, the solution was centrifuged and the absorbance of collected supernatant was measured at 540 nm. The percentage of hemolysis was calculated according to the following equation. The test was performed in triplicate.

\begin{array}{l} Percentage of hemolysis \\ = (A_{sample} - A_{negative control}) \\ \times 100 / (A_{positive control} - A_{negative control}) \end{array}

Cell line and culture conditions

The human osteoblastic cell line MG-63 and fibroblast cell line L929 (China Infrastructure of Cell Line Resource, Beijing, China) were used in the experiments. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were incubated in a humidified atmosphere with 5% CO₂ at 37 °C.

Cell viability assay

To determine the cytotoxicity of the matrix, the cell viability was assessed using an MTT assay. Briefly, each material was soaked in 1 mL of DMEM supplemented with 10% FBS and maintained in a humidified atmosphere with 5% CO₂ at 37 °C for 3 days. The eluate was collected and sterilized by passing through a 0.22 µm bacteria-retentive filter. L929 fibroblasts were seeded to a 96-well plate (10⁴ cells/well). After cell attachment for 24 h, the eluate of each group was added to the cells and incubated for 24 h. The controls were cultured in medium without eluate. Twenty microliters of the MTT solution (Sigma, US) was added and incubated at 37 °C for 4 h. DMSO (Sigma, USA) was then used to dissolve the resulting formazan and the OD value of the solution was measured at 570 nm.

Cell proliferation

MG-63 cells were seeded on the scaffolds at a density of 1 × 10⁴ cells per scaffold. First, 10⁴ cells suspended in 20 µl DMEM were seeded drop-wise on a scaffold in a 6-well plate. After incubation for 2 h in a humidified incubator, 4 mL of culture medium was added to each well. The cell culture medium was changed every 2 days. The cell proliferation was measured at desired time points (4h, 1, 3, 5 and 7 days) using a DNA Quantitation kit (Invitrogen). The cells were lysed in 100 µl distilled water using a freeze-thaw cycle. The cell lysate was added to 100 µl aqueous Hoechst 33258 in the TNE buffer from the kit. The fluorescence was measured with excitation and emission filters at 360 nm and 460 nm, respectively. The number of cells on the scaffold was quantified using a standard curved of known number of cells according to the manufacturer’s instructions.

Alkaline phosphatase activity assay

The alkaline phosphatase (ALP) activity was measured using an Alkaline Phosphatase Assay kit (Beyotime) according to the manufacturer’s protocol. After culturing for 3 and 7 days, the cell-scaffold complex was washed with PBS three times and the cells on the scaffold were homogenized in 100 µl cell lysis buffer (Beyotime). The lyses was centrifuged and the supernatant was transferred to a 96-well plate. The kit reagents were added in sequence and incubated for 30 min at 37 °C. This commercial kit was based on the concentration of colored p-nitrophenol produced by colorless p-nitrophenyl phosphate incubated with alkaline phosphatase at 37 °C. The absorbance was measured at 405 nm. The activity of ALP in the cells was normalized against the total protein content determined by the Bradford protein Assay Kit (Beyotime) and was expressed in nanomoles of p-nitrophenol produced per min per mg of protein (nmol/mg protein).

Animals and grouping

The maxillary sinuses of white New Zealand rabbits served as experimental models to evaluate the osteogenic potential of the NS-Sponge. The experiments were conducted according to the protocols (permit number: KQYY-201604–003) approved by the Committee on the Ethics of Animal Experiments of Beijing Stomatological Hospital.

A total of 24 male rabbits, weighing 2.5–3.0 kg, were chosen for this experiment. All the animals were individually housed during the experimental period, exposed to a light/dark cycle of 12:12 h, and provided with a standard laboratory diet and water ad libitum. After 1-week habituation, the 24 rabbits were equally distributed into 2 categories with a 4- and 12-week observation period, respectively. Within each category, 24 sinuses of 12 rabbits were elevated and randomly assigned to the following 4 groups depending on the materials implanted: autogenous blood clot (Cont) group (n = 6), gelatin sponge (Gelatin) group (n = 6), nanostructured sponge (NS-Sponge) group (n = 6), and Bio-oss collagen® (BO-Collagen) group (n = 6).

Surgery procedure and material implantation

Prior to the surgery, the animals were made to fast overnight. After an intramuscular injection of 0.3 mL Lumianning (Xylazine hydrochloride; Huamu, Jilin, China) to induce general anesthesia, preoperative hair removal was conducted in the nasal bone area. Primacaine Adrenaline (1:100000, Dentaires Pierre Rolland, France) was administered to the surgical site. The surgery was conducted according to the procedure described in a previous study.²⁷ A 2.5 cm vertical incision was made along the midline of the nasal dorsum, followed by flap creation in the skin and periosteum. Two bone windows, each with a diameter of 0.5 cm, were created bilaterally on the nasal dorsum, 2 cm anterior to the nasofrontal suture line, and 0.5 cm lateral to the midline (Figure 1). An elevator was used to separate the maxillary sinus mucosa from the bony wall and the bone substitutes were implanted. All the bone substitutes used in the animal surgeries were tailored into cylindrical shapes with a diameter of 0.5 cm and a height of 0.5 cm. In the Cont group, after the membrane elevation procedure, the elevator was used to support the membrane at an augmented height of 0.5 cm until the surrounding bony wall in situ filled the space with blood and the blood itself clotted. After implantation, the periosteum and skin were approximated with interrupted sutures. Antibiotics (Benzylpenicillin sodium, Shandong, China) were administered to prevent post-surgical infections.

Sequential cone beam computed tomography scan

To trace the mineralization of the augmented area and the augmented height transition of the materials implanted dynamically, cone-beam computed tomography (CBCT) scan (NewTom VG, Italy) was applied in each group of rabbits with a 12-week survival time. The technical parameters were 110 KV, 6 mA, 8 x 8 cm high-resolution FOV (voxel size 0.127 mm), and 5.4 s exposure time. The observation time points were before, immediately after, and four, eight, and twelve weeks after maxillary sinus floor elevation. As previously mentioned, these procedures were performed under general anesthesia. After the images were taken, all the data were imported onto the NNT Viewer (version 5.10, NewTom, Italy) to analyze the elevated height and bone mineralization. In this study, to measure the elevated height, all measurements were implemented at the sagittal plane along the top of the maxillary sinus, as shown in Figure 2. Augmented height was defined as the distance between the bony sinus floor and the sinus membrane.²⁷

Micro-computed tomography and histological examination

All the rabbits were sacrificed at predetermined time points via an overdose of intravenous anesthetics injected into the ear margin. The implanted area of the maxilla was dissected and fixed in 10% neutral buffered formalin for 14 days. All the formalin-preserved blocks were then transferred to 70% ethanol and viewed under a Micro-CT system (Inveon CT, Siemens, Germany) in high-resolution mode (pixel matrix: 3072 × 2048; slice thickness: 29 μm). The 3 D structure was then analyzed with the Inveon Workplace software version 2.2.0 (Siemens, Germany). The bone volume fraction (Bone volume/total volume, BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) were analyzed.

All the samples were then decalcified using 10% EDTA, embedded in paraffin, and cut into 5-μm-thick sections along the sagittal plane of the middle of the sinus. The sections were stained with hematoxylin-eosin (HE). These decalcified samples were further subjected to histological and histomorphometric observation and analysis. The histomorphometric measurements were performed using the Image-pro Plus software (Media Cybernetics, Inc, Rockville, MD, USA).

Measurements and analysis in micro-CT and histological findings

After carefully observing the Micro-CT and histological images of all samples, the measurements and analysis were carried out in two separate parts. First, the region from the basal bone of maxillary sinus, extending 1 mm into the sample in all groups was defined as the primary region of interest (ROI-1; Figure 3). Second, the entire augmented area of NS-Sponge and BO-Collagen group was defined as the second region of interest (ROI-2; Figure 3).

Since mineralized BO particles made it challenging to identify newly formed bone in micro-CT images, BV/TV, Tb.Th, and Tb.N were only analyzed in the Cont, Gelatin, and NS-Sponge groups in ROI-1. For the histomorphometric measurements, the percentage of new bone area in the ROI-1 of each specimen (new bone area/area of ROI) was assessed. In ROI-2, the percentage of new bone area and the remnant material area (remnant material area/area of ROI) in the NS-Sponge group and the BO-Collagen groups were quantified.

Statistical analysis

All the measurements in this study were performed blinded by the same evaluator. Mean and standard deviation values were presented in graphs. The SPSS software (version 20.0) was used for statistical analysis (*, P<0.05 was considered statistically significant). The homogeneity of variance and the normal distribution were analyzed for all dates. Statistical difference was detected using analysis of variance (ANOVA) and the Student–Newman–Keuls method was used to assess multiple comparisons.

Results

Characterization of surface morphological and mechanical properties

Surface micrographs provided details about the surface characteristics of the gelatin sponge, Bio-oss collagen®, and NS-Sponge. SEM images at low magnification showed that the gelatin and NS-Sponges possessed a microporous structure. At higher magnification, the gelatin sponge had smooth pore walls, while the nanostructured sponge showed a nanofibrous surface topography. Bio-oss collagen® was composed of inorganic bovine bone granules blended with collagen. As seen in Figure 4, the bovine bone particles were wrapped in cross-linked collagen fibers.

Figure 5 shows the resilience of the materials with and without compression. The nanostructured and gelatin sponges exhibited excellent resilience in recovering their original shape on removal of compression. BO-Collagen showed some deformation, however, barely rebounded after the compression was removed.

Hemocompatibility

As shown in Figure 6, all of the materials exhibited good hemocompatibility. A very low level of hemolysis, less than 5%, was detected in all groups, suggesting that the NS-Sponge, BO-Collagen and Gelatin would not result in severe hemolysis on the basis of ISO 10993–4:2002.

In vitro toxicity

The NS-Sponge, BO-Collagen, and Gelatin showed negligible cytotoxicity to L929 cells. The cell viability with the presence of different material extracts are shown in Figure 6. After incubation with the eluate of different materials for 24 h, the cell viability of all groups remained high and no statistical difference was detected among the groups.

Size variation, cell proliferation ability and ALP activity

The NS-Sponge showed a greater dimensional stability for maintaining cell growth. As shown in Figure 7, after co-cultured for 7 days, the cell/Gelatin and cell/BO-Collagen constructs significantly shrank whereas the size of the cell/NS-Sponge construct was maintained.

The cell number in each group was quantified by DNA content. The cell proliferation in the Gelatin, NS-Sponge, and BO-Collagen increased in a time-dependent manner. From Day 5, the cell number in the NS-Sponge group was greater than the other groups (p < 0.05).

ALP activity of MG-63 cells on the materials are shown in Figure 7. The Gelatin group exhibited significantly lower ALP activity than the other two groups both at Day 3 and Day 7 (p < 0.05). No significant difference was observed between the NS-Sponge and BO-Collagen at Day 3 and Day 7 (p > 0.05).

Cone-beam computed tomography examination

CBCT images were taken to trace mineralization in the sinus cavity, along with assessing the augmented height transition of the implanted materials (Figure 8). Immediately after the surgery, the implanted areas were radiolucent, displaying the soft-tissue density in the Cont, Gelatin, and NS-Sponge groups. On visual inspection at 4 weeks post-operatively in the NS-Sponge group, a little radiopaque area around the surrounding bony wall could be recognized. At 8 weeks, this opaque area was further extended, and at 12 weeks, it covered nearly the entire implanted area. The augmented membrane significantly collapsed in the Gelatin and Cont groups at 4 weeks, accompanied by decreasing augmented height, and both groups formed a thin layer of mineralized tissue adjacent to the sinus floor, which could be noted in the 8- and 12-week images. In the BO-Collagen group, a high-density image was seen at each time point. On account of the radiopaque BO granules, the mineralization process in BO-Collagen group could hardly be traced by CBCT.

Statistical results of the augmented height are presented in Figure 9. There was no significant difference in the augmented height among the groups immediately after surgery (p > 0.05). Nevertheless, at 4, 8 and 12 weeks postoperatively, both the Cont and Gelatin groups showed decreased augmented heights. Both these groups showed statistically significant differences compared to the NS-Sponge and BO-Collagen groups at 4, 8, and 12 weeks postoperatively (all p < 0.05). No significant difference was seen between the NS-Sponge and BO-Collagen groups (all p > 0.05).

Micro-CT measurements

The newly formed trabecular bone protruded from the surrounding bone wall and appeared as a white network in the Cont, Gelatin, and NS-Sponge groups. However, bright white radiopaque granules were seen in the implanted area in BO-Collagen (Figure 10A). Since the inorganic bovine bone mineral has similar or even higher density than the newly formed bone, the delineating trabecular bone in BO-Collagen group was not visualized. Therefore, the trabecular parameters were only analyzed in the Cont, Gelatin, and NS-Sponge groups. At 4 weeks, in the NS-Sponge group, the trabecular bone was formed adjacent to the surrounding bone walls. At 12 weeks, the mineralized tissue covered nearly the entire implanted area. In the Gelatin and Cont groups, the newly formed trabecular bone was restricted close to the bony sinus floor during the entire observation time. Considering the limited bone formation area of mineralized tissue in the Cont and Gelatin groups, the trabecular bone analysis was conducted in the above-mentioned ROI-1 area (Figure 3).

BV/TV, Tb.N, and Tb.Th increased in a time dependent manner in Cont, Gelatin and NS-Sponge (Figure 10B-D). The Cont group demonstrated significantly reduced BV/TV, Tb.N, and Tb.Th when compared to the other two groups at 4 weeks and 12 weeks post-operation (all p < 0.05). At 4 weeks, the BV/TV in Gelatin group was significantly higher than that of NS-Sponge group (p < 0.05). However, at 12 weeks, no statistical difference was detected between the Gelatin and NS-Sponge groups in terms of BV/TV, Tb.N, and Tb.Th (all p > 0.05).

Histological findings of new bone formation

Figure 11 shows the hematoxylin and eosin (H&E) staining results of new bone formation in the elevated sinus. Approximately 4 weeks after the implantation, the area of bone substitutes under the sinus membrane in the Gelatin and Cont groups contracted, with barely identifiable remnant material. The lifted membrane collapsed before complete healing and eventually led to the formation of new bone confined in a narrow space between the collapsed mucosa and the sinus bone wall at 12 weeks. However, in both the NS-Sponge and BO-Collagen groups, new bone ingrowth was seen specifically around the supporting biomaterials in the maintained space under the sinus membrane. At 4 weeks in the NS-Sponge group, the entire augmented area was almost filled with the scaffold and soft tissue. The newly formed trabecular bone surrounding the NS-Sponge could be distinguished at the marginal portion of the augmented sinus. At 12 weeks postoperatively, the NS-Sponge yielded consistent bone formation with time as the trabecular bone further extended, thus occupying the augmented area with a thicker and continuous structure. At both time points, large and irregular multinucleated giant cells were observed on the surface of the remnant NS-Sponge material. Interestingly, in the BO-Collagen group at 4 weeks, the new bone surrounding the BO particles was visible in most part of the augmented maxillary sinus, contrary to the NS-Sponge group, wherein the bone growth was marginal. Barely of such multinucleated giant cells could be identified in the 4- and 12-week BO-Collagen samples.

The histomorphometric analysis was performed at two regions of interest (ROI), because of the incoordinate osteogenic space under the sinus membrane among the groups (Figure 12). In ROI-1 (Figure 12(a)), the Cont group demonstrated significantly reduced percentage of new bone area as compared to other groups both 4 weeks and 12 weeks post-operatively (all p < 0.05). A significantly higher percentage of new bone area was observed in the Gelatin group than that in the other groups at 4 weeks (all p < 0.05). Meanwhile, no significant difference was seen between the NS-Sponge and BO-Collagen groups (p > 0.05). At 12 weeks, the percentage of new bone area in all tissue slides was increased compared to that in the previous four weeks in all groups. No significant difference was observed between the Gelatin and NS-Sponge groups (p > 0.05), while the percentage of new bone area was significantly higher in both these groups than in the BO-Collagen group (both p < 0.05).

In ROI-2, the histomorphometric analysis was performed throughout the entire implanted area under sinus membrane for the percentage of new bone area (Figure 12(b)) and the percentage of remnant material area (Figure 12(c)) in NS-Sponge and BO-Collagen groups. At 4 weeks after implantation, the BO-Collagen group (15.94 ± 1.03%) demonstrated a significantly higher percentage of new bone area than that of the NS-Sponge group (8.96 ± 1.24%) (p < 0.05). However, at 12 weeks, the NS-Sponge (28.56 ± 2.00%) group showed a significantly higher percentage of new bone formation as compared to the BO-Collagen group (23.56 ± 3.86%) (p < 0.05). The degradation of the bone grafts was quantified by measuring the percentage of the remnant material area. The average percentage of the remnant material area at 12 weeks remained lower than the remnant material at 4 weeks in both the NS-Sponge and BO-Collagen groups. Significant differences were seen between the two time points in the NS-Sponge group (p < 0.05), whereas no statistical difference was discovered in the BO-Collagen group (p > 0.05).

Discussion

Prior to clinical use, it is important to test bone substitutes through in vivo and in vitro preclinical studies. In our study, the typical gelatin sponge structure was modified using TIPS&P. Nanofiber formation was induced by phase separation, and pore size was controlled by porogen using a specific diameter. This NS-Sponge was tested in sinus floor augmentations for the first time and showed significant outcomes, suggesting a high potential for its future use in clinical practice.

The implanted materials should be biocompatible and integrate with the native tissue without causing cellular toxicity or hemolytic reaction. The cell viability and hemolysis results showed that the NS-Sponge, BO-Collagen, and Gelatin were safe with negligible toxicity. All the three materials sustained cell proliferation and osteoblast differentiation. The NS-Sponge and BO-Collagen might facilitate MG-63 differentiation towards a bone-like phenotype, as demonstrated by the ALP activity assay. The NS-Sponge had a better dimensional stability than the Gelatin and BO-Collagen, which would facilitate cell in-growth during tissue regeneration.

Some clinicians suggested that maxillary sinus augmentation is possible without grafting materials, in which case, blood clots act as the substitute.²⁸ Theoretically, using a patient’s own blood has some advantages, including exemption from extra bone substitutes, ease of application and access, and presence of growth factors in the blood clot.^29,30 Lundgren et al. suggested that merely elevating the sinus membrane, using endosseous implants as space maintainers, and facilitating blood clot formation can result in the formation of new bone.²⁸ Owing to this, a blood clot was used in our control group. BO-Collagen has gained wide acceptance as a bone substitute in numerous situations and is especially suggested in maxillary sinus floor augmentation.⁵ Therefore, rabbits implanted with BO-Collagen were categorized as the positive control group. The selected biomaterials exhibit diverse chemical and physical characteristics, some of which clearly demonstrate their advantages in maxillary sinus lift procedures.

Bone substitutes for a specific anatomical site, such as the maxillary sinus cavity, should possess geometric plasticity to fit into an irregular bone defect. Traditionally, clinicians choose granular bone grafts to fill the elevated sinus, including autogenous or allogenous/exogenous bone, or synthetic bone substitutes.³¹ When granular bone substitutes are implanted into a sinus cavity, especially through the narrow transcrestal osteotomy, surgeons often lose the control of the shape and size of the grafted materials. Each bulk-form material handled in this study was able to deform and be conveniently transported through a narrow approach.

In the case of maxillary sinus augmentation with bone grafts, the implanted material usually acts as a filler to support the space under the sinus membrane. However, the re-pneumatization of the maxillary sinus is shown to result in the adaptation of grafted materials in volume and shape.^32,33 In our study, we adopted the augmented height as the observation index to evaluate the space-maintaining capacity, which is usually used in clinical practice to assess bone gain.³⁴ CBCT is widely adopted as an ideal imaging technology in clinical use, as it noninvasively monitors bone formation in patients who receive sinus augmentation procedures.³⁵ In our study, the CBCT images showed that all the materials used were effective space maintainers between the sinus membrane and sinus floor immediately after surgery. However, in the 4-, 8-, and 12-week post-operative CBCT images, the sinus cavity exhibited significant re-pneumatization in the Cont and Gelatin groups that manifested as decreased augmented height. This may have been caused by the fast degradation of the gelatin sponge and fibrin network in the blood clot, thus not supporting new bone formation under the sinus membrane. Another reason may be the sinus membrane tendency to migrate toward the space supporter under air pressure.^36,37 As a result, only a thin layer of new bone was formed in the closed space under the collapsed sinus membrane in the Gelatin and Cont groups. This result was also confirmed in micro-CT and histological images. The low resorption rate of BO granules provides necessary volumetric stability to BO-Collagen in maxillary sinus augmentation.³⁸ In the present study, we found no difference in the augmented height between NS-Sponge and BO-Collagen during the 12-week observation period, indicating that the gelatin-based NS-Sponge could also maintain the space created under sinus membrane effectively enough to sustain bone formation.

The timing of titanium implant placement following one-stage bone grafting is a crucial aspect of successful implant treatment and this is always decided based on the radiographic evidence and the clinicians’ experience with bone graft healing. Kwon et al. measured series of grey values to analyze bone formation in BO grafts.³⁹ BO granules, however, exhibit a relatively high absorption of X-rays, which is consistent with the present study. Since BO granules and newly formed bone tissue exhibited low contrast in images, the grey values of BO and new bone could only be measured together.³⁹ However, this procedure needs extensive calculation, which makes it less intuitive in the clinic. In the NS-Sponge group, the mineralization process inside the scaffold was manifested as a gradually extending opaque area in CBCT images, which was different from the high-density radiographs in the BO-Collagen group. This radiographic change might provide clinicians with direct clues to assess bone remodeling in the defect.

Alayan et al. stated that native bone walls of a maxillary sinus have a positive impact on bone rebuilding.⁴⁰ Moreover, Busenlechner et al. noted that bone formation relates to the location in the graft material.⁴¹ Thus, it is reasonable to analyze tissue regeneration at a similar anatomic region. The occluded spaces in Cont and Gelatin groups resulted in an asymmetrical osteogenic space as compared to that in the NS-Sponge and BO-Collagen groups. Considering that the bone formation in Cont and Gelatin groups was confined to a 1 − 3 mm wide space under the sinus membrane, the area from the basal bone to 1 mm apically towards the sinus membrane was chosen as the region of interest (ROI-1) to analyze the trabecular bone patterns and the percentage of newly formed bone. In the BO-Collagen group, only histomorphological analysis was carried out to assess bone formation. This was because the mineralized BO granules in BO-Collagen are radiopaque, thereby hampering the evaluation of trabecular bone patterns on radiographic images; instead, decalcified sections allowed to clearly distinguish the bone tissue from the mineralized granules. Bone regeneration was observed in the augmented sinus filled with blood clot, as reported in previous studies.⁴² However, the blood clot group showed the least amount of bone formation compared to that of our other groups. In early stages of the healing process, the Gelatin group exhibited improved BV/TV and an increased percentage of newly formed bone. Following the 12-week healing period, the amount of regenerated bone was not different between the Gelatin and NS-Sponge groups but was significantly higher than that of the BO-Collagen group. Although satisfactory bone formation was observed locally near the basal bone with Gelatin, considering the bone height obtained in this study, gelatin sponge is recommendable only when a small amount of bone is required in the sinus cavity.

BO-Collagen and NS-Sponge exhibited superior space maintenance properties, and both materials showed satisfactory bone formation in histological images. Histomorphometric analysis assessing the entire augmented area (ROI-2) in BO-Collagen and NS-Sponge groups showed that, at 4 weeks, the newly formed bone was distributed only in the marginal zone of the graft near the basal bone in NS-Sponge, with the percentage of newly-formed bone being 8.96 ± 1.24%, which was significantly lower than that in the BO-Collagen group (15.94 ± 1.03%). The trabecular bone was distributed across the entire implanted material in the BO-Collagen group as per the H&E-stained images, contrasting with the marginal new bone formed at the NS-Sponge scaffold at 4 weeks. However, at 12 weeks postoperatively, the percentage of newly formed bone in NS-Sponge (28.56 ± 2.00%) was significantly higher than that in BO-Collagen (23.56 ± 3.86%).

In the current study, NS-Sponge was demonstrated to be a degradable biomaterial, evidenced by the analysis of remnant NS-Sponge pieces. By contrast, the remnant BO granules in BO-Collagen showed a slight decrease that no statistical difference was acquired during 4- to 12-week post-operation. Reportedly, residual BO particles can be detected till up to nine years after sinus augmentation in humans.⁴³ Some investigators state a similar performance of BO in the literature.^44,45 The low resorption rate of the biomaterials facilitates space maintenance in sinus augmentation; Perez-Sayans et al., however, indicate that the unabsorbed material possibly impedes substitution by the new bone.⁴⁶ This might explain the low percentage of newly formed bone in BO-Collagen at 12 weeks, despite the superior osteoconductivity demonstrated at the 4-week inspection.

Another noteworthy finding was that large multinucleated giant cells were noted on the surface of NS-Sponge, possibly related to the cell-mediated degradation of biomaterials.⁴⁷ On the contrary, BO-Collagen barely presented such cells in our study. These type of cells are considered to derive from macrophages fuse.⁴⁸ Different surface morphological and chemical clues of biomaterials influence macrophage phenotype, which in turn produces bioactive agents that induce a series of cellular responses.⁴⁹ In this study, the different histological features among the NS-Sponge and BO-Collagen suggest different degradation mechanisms of the two biomaterials, thereby warranting further in-depth investigations on the immunological response involved in NS-Sponge.

Conclusion

In this pre-clinical study, NS-Sponge effectively supported the sinus membrane in maxillary sinus augmentation. The biodegradable NS-Sponge enabled formation of a significantly higher percentage of new bone area compared to that formed by BO-Collagen at 12 weeks. The NS-Sponge possessed many other advantages, including excellent operability, cost-efficiency, and clear imaging characteristics. Further, plain gelatin sponges could be chosen when only a small amount of bone is required in maxillary sinus cavity. The clear superiority of NS-Sponge makes it a promising alternative biomaterial for clinicians to use during maxillary sinus augmentation. However, the future research should focus on the immunological response of NS-Sponge during tissue healing process.

Footnotes

Data accessibility statement

The data used to support the findings of this study are available from the corresponding author upon request.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Beijing National Science Foundation [grant number 7172088]; and the National Natural Science Foundation of China [grant number 81974153].

ORCID iD

Pan Ma

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