Compression-resistant polymer/ceramic composite scaffolds augmented with rhBMP-2 promote new bone formation in a non-human primate mandibular ridge augmentation model

Abstract

Purpose

This study was designed to test the hypothesis that compression-resistant (CR) scaffolds augmented with recombinant human bone morphogenetic protein-2 (rhBMP-2) at clinically relevant doses in a non-human primate lateral ridge augmentation model enhances bone formation in a dose-responsive manner without additional protective membranes.

Methods

Defects (15 mm long x 8 mm wide x 5 mm deep) were created bilaterally in the mandible of 9 hamadryas baboons. The defect sites were implanted with poly(ester urethane) (PEUR)/ceramic CR scaffolds with 0.75mg/ml rhBMP-2 (CR-L), 1.5mg/ml rhBMP-2 (CR-H), or control CR scaffolds without rhBMP-2 (CR). The primary outcome of ridge width and secondary outcomes of new bone formation, cellular infiltration, and integration with host bone were evaluated using histology, histomorphometry, and μCT at 16 weeks following implantation.

Results

New bone formation in the mandible was observed in a dose-responsive manner. CR-H promoted significantly greater new bone formation compared with the CR group. In all groups, ridge width was maintained without an additional protective membrane.

Conclusions

CR scaffolds augmented with a clinically relevant dose of rhBMP-2 (1.5mg/ml) promoted significant new bone formation. These results suggest that a compression-resistant PEUR/ceramic composite scaffold without a protective membrane may be a potential new rhBMP-2 carrier for clinical use.

Introduction

Dental implants are frequently used to restore dentition. However, mandibular reconstruction is often necessary to enhance bone volume prior to dental implants. A number of materials have been utilized for ridge augmentation, but autograft remains the gold standard. However, complications associated with autograft including donor site morbidity, limited quantity, poor vascularization, and potential failure to integrate with host bone preclude its use.^1,2 These limitations have led to the development of scaffolds augmented with growth factors for ridge reconstruction.^3,4

Bone morphogenetic proteins (BMPs) were first discovered as osteoinductive growth factors by Urist.⁵ Of the many known BMPs, BMP-2 plays a critical role in bone formation.⁶ Processes have been developed for the manufacture of recombinant human BMP-2 (rhBMP-2) and its subsequent clinical use.⁷ Absorbable collagen sponge (ACS) rhBMP-2 carriers are an FDA-approved treatment (INFUSE^® bone graft, Medtronic) for posterior-lateral spinal fusion, tibial fractures, sinus lift procedures, and extraction socket defects.⁸ However, the ACS carrier is unable to withstand compressive forces and requires the use of either resorbable (collagen, polylactic acid, polyglycolic acid) or non-resorbable membranes (e-PTFE, titanium mesh). Both resorbable and non-resorbable membranes have limitations including wound dehiscence and healing complications.⁹ Additionally, resorbable membranes cause swelling and inflammation concerns while non-resorbable membranes must be subsequently removed.¹⁰

The successful use of rhBMP-2 in ridge augmentation is dependent on both the properties of the carrier and protein concentration.¹¹ The ideal carrier is resorbable and optimizes predictable bone formation through controlled release of rhBMP-2 and space maintenance. Resorbable lysine-derived poly(ester urethane) (PEUR) scaffolds are promising carriers that have been frequently utilized for bone grafting applications.^12,13 The addition of ceramic particles to PEUR scaffolds has shown improved mechanical properties and osteoconductivity.¹⁴ Compression-resistant (CR) PEUR/ceramic composite scaffolds have previously been shown to enhance bone formation in femoral condyle plug defects in sheep.¹⁵

In both a canine mandibular saddle defect¹⁶ and lateral ridge defect¹⁷ model, rhBMP-2-augmented CR PEUR/ceramic composite scaffolds promoted new bone formation without the use of a membrane. However, the demonstration of bone formation utilizing CR composite carriers augmented with clinically relevant rhBMP-2 doses in primates is essential prior to its use in humans.¹⁸ The purpose of this study was to test the hypothesis that CR composites without membranes maintain ridge width and promote new bone formation in a dose-responsive manner in a non-human primate model. CR scaffolds incorporating clinically relevant doses (0, 0.75, or 1.5 mg/ml) of rhBMP-2 were evaluated in a non-human primate lateral ridge augmentation model. The primary outcome of ridge width and secondary outcomes of new bone formation, cellular infiltration, and integration with host bone were evaluated with histology, histomorphometry, and μCT at 16 weeks post-operatively.

Materials and Methods

Materials

Lysine triisocyanate (LTI)-poly (ethylene glycol) (PEG) prepolymer was acquired from Ricerca Biosciences LLC. Glycerol, ε-caprolactone, stannous octoate, APTES triethylene diamine, and dipropylene glycol were purchased from Sigma-Aldrich (St. Louis, MO) and utilized for polyester triol synthesis. Glycolide and DL-lactide were also used in the polyester triol synthesis and were purchased from Polysciences (Warrington, PA). Medtronic Spinal (Memphis, TN) supplied rhBMP-2 and ceramic (CM) (15% hydroxyapatite, 85% beta tricalcium phosphate) granules. CM granules ranged from 100–500 μm for improved handling properties.

Polyester triol synthesis

The polyester triol was synthesized following a previously published method.^19,20 A 10% (w/w) solution was made using triethylene diamine and dipropylene glycol. Briefly, glycerol, ε-caprolactone, glycolide, and DL-lactide monomers were mixed for 40h under argon at 140°C to yield a viscous fluid. The resulting fluid was hexane-rinsed (3X) and dried under vacuum at 80°C for 48 h. The synthesized polyester had a molecular weight of 450 g mol⁻¹, and the backbone consisted of 70% ε-caprolactone, 20% glycolide, and 10% DL-lactide.

Compression-resistant (CR) scaffold fabrication

The CR scaffolds were fabricated as described previously.¹⁷ Prior to the study, CR scaffold materials were gamma-irradiated using a dose of 25 kGY. Immediately prior to scaffold implantation within the defect, CM particles (45 wt%), lyophilized rhBMP-2, polyester triol, triethylene diamine (1.1 pphp), and LTI-PEG were mixed for 60s by hand in a 5-mL mixing cup. The NCO:OH index was set at 115 (15% excess NCO). An index of 115 produces a sufficiently cured foam and was selected to account for additional OH groups arising from the presence of blood and water within the defect space upon injection of the reactive scaffold.²¹

Animals

Utilizing the ridge width outcomes from our previous canine study¹⁷, a power analysis for this non-human primate study was performed with an assumed effect size of >0.78, a power of 80%, and alpha of 0.05 to determine the sample size of 6 per group. Therefore, 9 papio hamadryas baboons (two implants per animal) were used to test the hypothesis that CR-H promotes the highest ridge width. This number of animals is consistent with previous ridge augmentation studies evaluating rhBMP-2 in non-human primates.^22–24 Skeletally mature males and females (5 males, ~23 kg, and 4 females, ~15 kg) of average weight were selected. The Institutional Animal Care and Use Committee of the Mannheimer Foundation and the ACURO of the US Army approved this study. Surgical and care procedures were performed in compliance with the Animal Welfare Act, Animal Welfare Regulations, and the Guide for the Care and Use of Laboratory Animals.

Pre- and postoperative care

Prior to and following surgical procedures, the animals were kept in separate cages. All surgical procedures were performed under general anesthesia in a sterile operating room. Animals were given antibiotic ampicillin (5 mg/kg) and sustained release opioid buprenorphine (0.2 mg/kg) prior to surgery. An intravenous injection of ketamine (10 mg/kg) and glycopyrrolate (0.004 mg/kg) were used to induce general anesthesia, and an endotracheal tube was placed to maintain general anesthesia with isoflurane (approximately 0.5–4%) in oxygen to effect. Postoperatively, antibiotic enrofloxacin (5mg/kg) and meloxicam (0.1 mg/kg) for pain control were administered via intramuscular injection and subcutaneously, respectively, for 7 days. Additionally, extraction sites were inspected and flushed with Nolvadent. Following the procedures, the animals received soft food twice daily and had ad libitum access to water.

Surgery 1 (Extraction and Defect Creation)

To prepare the alveolar ridge defect, incisions were made by full mucoperiosteal flap reflection. A separating disk was used to ease root extraction of all two-rooted teeth prior to removal. The buccal bone plate was removed to create a defect measuring approximately (length 15 mm, height 8 mm, depth 5 mm), and the defect margins were outlined utilizing a small round burr. Sterile saline irrigation was performed with all drilling. Flaps were closed with sutures. Each animal obtained bilateral defects.

Surgery 2 (Ridge Augmentation)

The extraction sites were accessed and debrided after a 3-month healing period. All granulation tissue was removed from the defect site, and defect margins were redefined to ensure that the defects measured approximately 15–16 mm mesiodistally, 8–9 mm apico-coronally, and 5–6 mm bucco-lingually (Figure 1A). Lyopholized rhBMP-2 at no (0.0 mg/mL), Low (0.75 mg/mL), or High (1.5 mg/mL) dose was mixed with the individual components of the CR scaffolds (n=6 per group) and injected into the defect site (Figure 1B). Prior to suturing, the CR grafts cured within the defect space for roughly 10 min (Figure 1C–D). The primates received randomized animal IDs to randomize treatment groups such that each animal received a different treatment group in each defect.

Figure 1.

Surgical images. (A) Surgery to create lateral ridge defect. (B) CR immediately following injection into defect site. (C) Cured CR graft 10 minutes after injection. (D) Wound closure following graft placement.

Sacrifice

All animals were euthanized 16 weeks after the second surgery with an intravenous injection of pentobarbital Sodium (Fatal Plus^®) (100 mg/kg body weight). The mandibles were extracted and formalin (10%) fixed for two weeks.

μCT Analysis

After extraction, the fixed mandibles were analyzed using a μCT50 (SCANCO Medical, Basserdorf Switzerland). Scans were performed at 70 kVp energy, 200 μA source current, 1000 projections per rotation, 800 ms integration time, and an isotropic voxel size of 17.2 μm. 3D reconstructions of the defect were generated from Scanco evaluation software in which a 100-slice region within the middle of the defect was contoured to quantitatively assess bone formation. The total volume of new bone within the defect region was measured to calculate bone volume fraction (BV/TV).

Histology

Bone regeneration was assessed via non-decalcified histology.²⁵ After formalin fixation, samples were dehydrated with gradients of ethanol. Samples were then embedded in poly(methyl methacrylate) and allowed to polymerize for 7 days. Sections were cut from the center of each defect (bucco-lingually) with an Exakt band saw. Polished ground sections (<100μm) were stained with Sanderson’s rapid bone stain to qualitatively assess cellular infiltration and bone regeneration.

Histomorphometry

Metamorph software (Version 7.0.1, Waltham, MA) was used to quantitatively analyze ridge width and new bone formation in the histological sections. Ridge width was measured as a function of height at the mid-section of the defect. The ridge was measured from the back wall of the defect in 2-mm increments from the coronal base of the defect and subsequently normalized to the width of the coronal base of the defect. The area of interest (AOI) for new bone formation was defined as a rectangle measuring 8 × 5 mm, corresponding with the initial defect size, where the AOI aligned with the sagittal wall and coronal base of the defect. New bone (red) and infiltrating cells (blue) were quantified within the AOI of each section utilizing Metamorph. Host bone integration, characterized by the amount of new bone formation along the host bone interface at the sagittal wall of the defect, was evaluated by measuring the amount of new bone extending into the defect space over a 1-mm distance along the sagittal wall of the defect.

Statistical Analysis

Summary statistics including mean and median values were calculated for each defect. The μCT and histomorphometric parameters were plotted as mean ± standard deviation and analyzed using a non-parametric Kruskal-Wallis test with Dunn’s tests for pairwise comparisons by GraphPad Prism 8.0 software (GraphPad Software). The p-values are reported for significant (p < 0.05) differences between groups.

Results

Surgical outcomes

The surgical procedures were uneventful. However, certain limitations exist when using non-human primates.²⁶ Despite pain care and monitoring, three grafts were removed by the primates between days 6–9 following the second surgery. These samples were excluded from analysis. Between days 9–14 post-op, some of the primates began to pick at their sutures, resulting in exposure of the implants. The presence or absence of the implants could not be confirmed from radiographs taken between days 14–21, and thus the possibility that the implants were either partially or fully removed by the animals cannot be excluded. However, all wounds fully healed and no subsequent infections arose. One animal presented an abnormal defect in the right side of its mandible discovered upon the first surgical procedure. This sample was not included in the analysis. Ultimately, 14/18 samples were used for analysis.

μCT Analysis

μCT images obtained after 16 weeks show differences in healing between the treatment groups (Figure 2A). Representative images of the coronal planes revealed new bone present in all three groups. A significant difference in BV/TV (Figure 2B), calculated from 3D reconstructions of the mandible after 16 weeks, was observed between the CR-H group (83.83 ± 0.07 %) when compared with the CR group (59.31 ± 0.10 %) (Table 1).

Figure 2.

μCT analysis of new bone formation. (A) Representative μCT images of the mandible after 16 weeks. (B) Bone volume/ total volume (BV/TV) after 16 weeks for each of the treatment groups.

Table 1.

Volumetric measurements of BV/TV by μCT at 16 weeks.

Treatment Group		BV/TV
CR	Mean ± SD	0.59 ± 0.10
	Median	0.58
CR-L	Mean ± SD	0.67 ± 0.14
	Median	0.68
	p	>0.999
CR-H	Mean ± SD	0.84 ± 0.07*
	Median	0.84
	p	0.0264

^*Significantly different compared with CR group using Dunn’s test (p<0.05).

Histological analysis

Samples stained with Sanderson’s rapid show new bone (red), cells (blue), and residual CM (black). The mandibles of the animals varied in overall volume due to the inclusion of both males and females in this study; however, the defect size and AOI were standardized in all animals regardless of sex. No evidence of inflammation was present within the defect space. New bone formation was seen in all groups, especially along the outer ridge (Figure 3A), but samples from the CR-H group exhibited the most uniform healing within the defect. High-magnification images (Figure 3B) showed new woven bone and new lamellar bone indicated by concentric lamellae. The CR group displayed more woven bone versus lamellar bone seen in the CR-L and CR-H groups.

Figure 3.

Representative images of histological sections. (A) Low- images (4X, 2mm scale bar) and (B) high-magnification (10X, 500μm scale bar) images display new bone (red), infiltrating cells (blue), and ceramic particles (black). Woven and lamellar bone is indicated by blue and yellow arrows, respectively.

Histomorphometry

The amount of new bone formation within the defects treated with CR, CR-L, and CR-H implants is listed in Table 2. Similar to the μCT results, histomorphometric analysis showed significant new bone in the CR-H group (68.3%) compared with the control group (43.9%) (Figure 4A). Comparatively, less new bone was seen in both the CR-L and CR groups. Cellular infiltration trended higher in both the CR-L and CR groups compared with the CR-H group but the differences were not significant (Figure 4B). Integration of host bone with the implant was assessed by measuring the area% new bone near the periphery of the implant (1 mm distance from the sagittal wall of the defect). The area% new bone near the periphery was 55 – 82% (Figure 4C), which indicates that new bone grew into the implant resulting in its integration with the host bone. Ridge width as a function of ridge height was measured at 0, 2, 4, and 6 mm above the baseline of the defect (Table 3) for each sample. Ridge width was maintained in all of the groups (Figure 4D).

Table 2.

Area% (bone) and Area% (infiltrating cells) at 16 weeks measured histomorphometrically.

		Area% Bone	Area% Cells
CR	Mean ± SD	43.94 ± 5.45	13.31 ± 4.12
	Median	53.38	13.18
CR-L	Mean ± SD	56.88 ± 12.3	17.31 ± 9.98
	Median	59.18	14.62
	p	0.3256	>0.999
CR-H	Mean ± SD	68.27 ± 8.19*	7.83 ± 4.01
	Median	66.99	7.94
	p	0.0337	0.6147

^*Significantly different compared with CR group using Dunn’s test (p<0.05).

Figure 4.

Histomorphometric analysis. (A) Histomorphometric analysis of new bone (red) and (B) infiltrating cells (blue) within the area of interest. (C) Histomorphometric analysis of integration of host bone with the implants. The area% new bone near the periphery of the implant (1 mm distance from the sagittal wall of the defect) was measured. (D) Normalized ridge width within the area of interest at 0, 2, 4, and 6 mm from the baseline of the defect.

Table 3.

Ridge width measured at 16 weeks at 0, 2, 4, and 6, mm above the base of the defect. Ridge width was normalized to the baseline.

		0	2	4	6
CR	Mean ± SD	1.00	0.99 ± 0.10	0.90 ± 0.19	0.75 ± 0.19
	Median		1.00	0.92	0.74
CR-L	Mean ± SD	1.00	1.03 ± 0.07	1.00 ± 0.06	0.85 ± 0.08
	Median		1.04	0.99	0.84
CR-H	Mean ± SD	1.00	1.07 ± 0.09	0.99 ± 0.15	0.82 ± 0.16
	Median		1.09	1.05	0.89

Discussion

This study evaluated bone regeneration in CR scaffolds augmented with rhBMP-2 in a pre-clinical non-human primate lateral ridge augmentation model. Three concentrations of rhBMP-2 (0, 0.75, and 1.5 mg/ml) were used to assess ridge width and new bone formation in response to the rhBMP-2 dose.²⁷ We hypothesized that the CR-H (1.5 mg/ml) carrier would enhance ridge width, which was the primary outcome in the study, compared with the CR-L (0.75 mg/ml) or control (0 mg/ml) groups. Although there was not a significant increase in ridge width with the CR-H carrier augmented with the same concentration of rhBMP-2 as the FDA-approved INFUSE^® bone graft, significantly higher BV/TV (Figure 2) and area% bone (Figure 4) in the lateral ridge defects were observed compared with CR samples. A post-hoc BV/TV effect size of 1.922 was calculated utilizing a Kruskal-Wallis H test based on the experimental data and actual number of replicates included in each group, which provided a power of >99%.

rhBMP-2 has previously been used to promote mandibular reconstruction in non-human primate preclinical models.^18,28,29 In this study, the non-human primate was chosen because of its anatomic and biological similarity to humans. Additionally, non-human primates experience the same dose-limiting toxicity of humans³⁰, and non-human primate studies are required by regulatory agencies to demonstrate the safety and efficacy of potential new therapies.³¹ These characteristics render the non-human primate lateral ridge augmentation model valuable for evaluation of potential new periodontal regenerative therapies.

Membranes are frequently used in ridge augmentation to provide a barrier that prevents soft tissue ingrowth while maintaining space within the defect to allow for new bone formation.³² They must remain covered for the entirety of the healing period as early membrane exposure has been shown to lead to overall negative clinical outcomes.^33–35 Despite the frequent use of membranes, some studies have suggested no difference or increased new bone formation in grafts without membranes compared to those with membranes.^36,37 The ceramic granules impart compression-resistant mechanical properties to the PUR/ceramic scaffolds.^16,28 At the same dose of rhBMP-2 delivered to lateral ridge defects in hounds, ridge width and new bone formation at 16 weeks in CR carriers with no membrane was comparable to that in the collagen carrier with a membrane.¹⁷ Consequently, in an effort to reduce the number of animals, we did not include a collagen with membrane group in the non-human primate study.

The carrier for rhBMP-2 is crucial in the induction of new bone. CR composites contained 45 wt% CM particles. The ceramic particles were added due to their osteoconductive and CR properties as reported previously in porcine ²⁸, sheep¹⁵, and canine^16,17 models. Residual CM particles were observed in some sections but could not be reliably quantified due to their low concentration. Resorption of CM particles was also observed in previous studies evaluating CR carriers for rhBMP-2 in canine ridge augmentation models.^16,17 These observations are consistent with a previous study reporting that CM granules implanted in extraction sockets in human patients were infiltrated and resorbed by osteoclasts.³⁸ Histomorphometric analysis showed preserved anatomic contour of the ridge in all groups, which contrasts with our previous study reporting that space maintenance in CR carriers increased with rhBMP-2 concentration in a canine-model of ridge augmentation.¹⁷ These findings suggest that CR carriers provide space maintenance and prevent tissue collapse into the defect site.

Considering that rhBMP-2 increases recruitment of osteoprogenitor cells³⁹, we assessed cellular infiltration into the implants by histomorphometry. The area% infiltrating cells and granulation tissue within the defect space at 16 weeks trended higher in the CR and CR-L groups compared with the CR-H group. However, differences in cellular infiltration between groups were not significant. This finding suggests that although overall cellular infiltration is comparable between groups, a larger percentage of cells in the CR-H group are osteoprogenitor cells that promote new bone formation.

This study evaluated clinically relevant doses of rhBMP-2 comparable to those used in human patients. While we cannot exclude the possibility that some of the implants were partially or completely removed at 9–14 days due to graft exposure, we have previously reported that the CR carriers deliver a 25% burst release of rhBMP-2 within the first 3 days and ~70% release after 14 days.¹⁷ In contrast to the adsorption-controlled release reported for the collagen carrier⁴⁰, the release of rhBMP-2 from CR carriers is diffusion-controlled.¹⁷ Thus, most of the rhBMP-2 was released from the CR grafts at the time of exposure. Consistent with a previous non-human primate study utilizing rhBMP-2 coated titanium implants⁴, a dose-responsive increase in new bone formation was observed amongst groups. CR-H specimens demonstrated increased new bone formation compared with the CR group. Although ridge width was maintained in all groups, CR and CR-L showed less new bone formation near the periphery of the implant compared with the CR-H group. These observations suggest that CR carriers for rhBMP-2 promote new bone formation within the lateral ridge when administered at the same dose as the FDA-approved INFUSE^® bone graft.

To minimize the number of animals needed to maintain statistical power, the study did not include multiple time points or an empty defect as a negative control, and thus the extent of self-healing of the defects could not be assessed. Despite the fact that the defect was critical-size^18,41,42, new bone formation in the control group without rhBMP-2 was observed. The observed new bone formation in the CR group is consistent with previous studies reporting modest new bone formation utilizing a compression-resistant carrier without rhBMP-2 in a non-human primate ridge models.^26,28 However, in both of these previous studies, the groups with rhBMP-2 showed increased new bone formation compared with CR carriers without rhBMP-2, suggesting that the addition of rhBMP-2 enhances the reproducibility of bone regeneration in non-human primate models. Another study utilizing a non-compression-resistant polylactic-co-glycolic acid foam carrier without rhBMP-2 in a non-human primate model showed negligible new bone formation in an empty defect.¹⁸ While these findings suggest that space maintenance provided by compression-resistant scaffolds without rhBMP-2 may be sufficient to stimulate new bone formation, the addition of rhBMP-2 is anticipated to promote more predictable bone healing.

This study showed that local delivery of 1.5 mg/ml rhBMP-2, which is the concentration used clinically with a non-compression-resistant collagen carrier, increased new bone formation but not ridge width at 16 weeks. A limitation of the study is that rhBMP-2 may have accelerated ridge augmentation, which would require assessment of outcomes at earlier (e.g., 8 weeks) time points. Enhanced new bone formation at 16 weeks is not necessarily an indicator of the ability of the newly formed bone to support functional loading of dental implants, which is the primary clinical endpoint for lateral ridge augmentation procedures. Future studies will assess the ability of CR carriers to regenerate sufficient new bone to support restored dentition.

Conclusion

In a clinically relevant non-human primate model, we tested the hypothesis that CR PEUR/ceramic carriers for rhBMP-2 promote new bone formation in a dose-responsive manner. CR scaffolds maintained comparable ridge width in all groups and promoted new bone formation in the expected dose-responsive manner. Based on the results of this study, CR PEUR/ceramic bone grafts may be effective new carriers for rhBMP-2 for clinical use in lateral ridge augmentation procedures.