Ridge Preservation Using a Novel Freeze-dried Enzyme-deantigenic Bone Paste: A Histomorphometric-retrospective Pilot Case Series

INTRODUCTION

Tooth extraction is usually followed by ridge remodeling and resorption, because of the lack of masticatory load, according to a well-known spatial and temporal pattern.^1–3 Atrophy may progress to such an extent that implant placement may become unfeasible; even when it can be carried out, any implant-supported rehabilitation may be at risk of functional and esthetic failure.² Ridge atrophy may be contrasted by grafting the post-extractive sockets with a bone graft according to the ridge preservation⁴ technique. Autogenous bone is still considered as the gold standard concerning bone grafting, because of the cells’ osteoinductive effect and the growth factors it contain.⁵ Autogenous bone collection requires opening a second surgical site, either intraoral or extraoral, thus exposing the patient to additional risk and discomfort.⁶ Several synthetic and natural biomaterials have been proposed as autogenous bone substitutes.⁷ Xenografts, animal-derived bone grafts, may represent a feasible option as bones of all mammal species, including mankind, share a similar three-dimensional (3D) morphology and chemical composition of their mineral portion.⁸ Hence, xenografts might have a biological advantage over other natural or synthetic grafts when used as bone grafts to regenerate the patient’s bone.⁹ So far, the process used to make non-antigenic animal bone seems to strongly affect the remodeling characteristics of the graft. Recent studies, in fact, have shown that the enzyme-deantigenic equine bone (EDEB) displays a different behavior than the anorganic bovine bone (ABB), the most used xenograft in oral and maxillofacial surgery, with EDEB undergoing significantly faster remodeling and producing, at a given time, a greater amount of the newly formed bone.^10,11 To manufacture ABB, bovine bone is subjected to high temperatures to eliminate bovine antigens;¹² EDEB, instead, is made non-antigenic subjecting equine bone to the action of hydrolytic enzymes.⁹ These latter allow the preservation of the 3D structure of the mineralized bone and the conservation of the natural bone collagen in native conformation. EDEB is being used extensively in different fields, including oral, maxillofacial, and orthopedic surgery.^13–22 Biomaterials for ridge preservation should be easy-handling and should display a certain degree of space-keeping properties. Furthermore, they should favor soft tissue regeneration when they are grafted in post-extractive sockets, and gingival healing is achieved by second intention.²³ Bone pastes fulfill these requirements, as they are user-friendly formats that are easy to handle and capable of holding optimal space-keeping properties when they are hard enough (e.g., when they are moldable). Moldable pastes can adapt to the site they are grafted in, assuring complete socket filling and direct contact with the surrounding bone tissue, possibly facilitating bone repair.²⁴ Recently, a novel line of bone pastes has been placed on the market as the evolution of EDEB and can be indicated as EDEBEX. This product consists of a hydrogel (namely Exur®, from which the name EDEBEX: EDEB + Exur) made by polyethylene glycol/hydroxyl-propyl methyl cellulose (PEG/HPMC)-based that acts as a carrier, containing cancellous and cortical EDEB granules and/or equine demineralized bone matrix.²⁵ The gel is added with a subsidiary amount of vitamin C modulating its rheology. When human bone marrow stem cells were co-cultivated with EDEBEX, they were found to over-express some bone regeneration modulators, such as, the RUNT-related transcription factor 2 (RUNX2), the bone sialoprotein, and the osteocalcin.²⁵ When EDEBEX was grafted in artificially induced femoral defects in rabbits, histological assessment of the regenerated bone after 1 and 2 months showed that the bone tissue had undergone a significant re-organization within the lesion boundaries.²⁵ At present, clinical and histological data concerning the use of EDEBEX in humans are limited to a recent case report with respect to one patient who underwent socket preservation and was followed up for 36 months.²⁶ The patient underwent successful implant-supported bone rehabilitation; at 36 months, the peri-implant bone levels had been maintained with the implant being successful according to the Albrektsson and Zarb criteria;²⁷ histomorphometric analyzes showed that the amount of the newly formed bone at implant insertion, 3.5 months after the grafting surgery, was 60.12%. The authors of the present study have been using a freeze-dried version of EDEBEX for some months and have collected additional clinical and histomorphometric evidence concerning its use to achieve ridge preservation. This small case series’ aim is to describe these additional results.

MATERIALS AND METHODS

This case series concerns three patients who underwent socket preservation surgeries at the author’s facility. The first patient (Patient A, 70 years old, male) was treated because of a fractured implant (3.6) and the adjacent irrecuperable tooth (3.5), both requiring removal; the second patient (Patient B, 58 years old, female) had one tooth (4.5), already devitalized and supporting a bridge, extracted because of an invasive caries; and the third patient (Patient C, 36 years old, female) had residual roots (1.6) from a destructing caries that required removal. All patients were treated according to the principles of ridge preservation, and all had the freeze-dried equine-derived bone paste EDEBEX (Activabone Putty, Bioteck S.p.A., Arcugnano, Italy) grafted in their post-extractive sockets. Radiographs and clinical pictures of the three patients and the surgeries they were subjected to are shown in Figures 1 to 3, respectively. In patients A and C, the grafted site was protected with a collagenic 3D matrix (Biocollagen Xenomatrix, Bioteck S.p.A., Arcugnano, Italy). In patient B, instead, neither a membrane nor a collagenic matrix was used to cover the graft. Healing by second intention was achieved in patients B and C. For all three patients, a two-step rehabilitating procedure was planned, involving grafting the sockets to preserve them from resorption followed by a delayed implant placement. Patients provided their informed consent to the treatment and to the collection of the biopsy for research publication purposes.

All patients underwent a thorough oral hygiene 2 days before the surgery. For antibiotic prophylaxis, 2 g of amoxicillin/clavulanic acid (Augmentin, GlaxoSmithKline, Verona, Italy) were administered 1 hour before the surgery and then every 12 hours for 8 days. The patients also rinsed their mouth for 2 minutes with chlorhexidine 0.20% mouth rinse (Corsodyl, GlaxoSmithKline) and received 100 mg of a non-steroidal anti-inflammatory drug (Aulin, Roche, Milano, Italy). Local anesthetic was injected into the oral mucosa with 1% articaine with epinephrine 1:100,000 (Molteni Dental, Milano, Italy).

The teeth were extracted atraumatically and the sockets debrided from any residual of the fibrous tissue. Subsequently, the bone paste — still dry — was placed into the sockets and gently pressed with a round instrument to fill them. A flap was elevated only for patient A. After grafting, a 3D collagenic matrix was used to cover the graft in patients A and C. In patient A, the flap was sutured with single stitches, while gingival margins of patients B and C were stabilized using cross stitches, and gingival rims were left open seeking for second intention healing. A 4−0 non-resorbable suture (Vicryl Plus, Ethicon, Johnson and Johnson, Pomezia, Italy) was used in all cases. A second surgery was performed after 4 months in patient A, after 6 months in patient B, and after 7 months in patient C. In all cases, the clinical appearance of soft tissues and the radiographic appearance of the grafted area were quite satisfying. Antibiotic prophylaxis, postsurgical treatment, anesthetic treatment, and pain management were carried out similar to the first surgery. After gaining access to the bone ridge, a bone core (patient A, position 3.5; patient B, position 4.5; patient C, position 1.6) was collected using a trephine and placed in a test tube containing buffered 10% formalin for subsequent histological analysis. The implant sites were then prepared following the drilling sequence suggested by the manufacturer and the implants were placed (patient A, position 3.5, 3.7 × 10 mm; Safe, Biotec S.r.l., Dueville, Italy; patient B, position 4.5, 3.7 × 8.0 mm; position 4.7, 3.7 × 8.0 mm, Safe, Biotec S.r.l.; patient C, position 1.6, 4.1 × 10 mm, Safe, Biotec S.r.l.). Patient A was later rehabilitated with a four-element monolithic-zirconia bridge supported by the implant placed in position 3.5 and the two implants still in place in positions 3.7 and 3.8. Patient B was rehabilitated with a three-element monolithic-zirconia bridge supported by implants in positions 4.5 and 4.7. Owing to personal reasons, patient C decided to postpone the rehabilitation with a single monolithic crown supported by the implant in position 1.6. Patients were followed up every month for the next 6 months, and then every 6 months after implant surgery.

RESULTS

All patients healed uneventfully, and their final rehabilitation was both functionally and esthetically satisfactory. At the last follow-up, all prostheses were working effectively, and no gingival recessions could be observed. The marginal bone loss for all implants placed into the regenerated sockets was within the success threshold defined by Albrektsson and Zarb.²⁷

DISCUSSION

No doubt exists, when reading the literature published on ridge preservation, that ridge preservation procedures indeed result in clinically significant less horizontal and vertical ridge bone loss, even if a certain degree of bone remodeling cannot be, however, avoided.³⁰ It is also known that appropriate flap management and the use of barrier membranes favorably contribute to bone preservation.³¹ Yet, questions still exist concerning the surgical procedure, the biomaterial, or the combination of the two that should be preferred.^32–34 In 2015, a Cochrane meta-analysis concerning several randomized clinical trials and more than 200 extraction sites concluded that different grafting procedures and biomaterials did not show any clinically significant difference and called for more clinical research on the subject.³⁵ The results of the present retrospective pilot case series study show that the dry paste EDEBEX used provided good clinical and histomorphometric results. Our data may be regarded as fairly in line with those of the recently published case report by Di Stefano et al.²⁶ where the amount of the newly formed bone was about 60%. In our study, we observed a slightly higher amount of mineralized matrix (i.e., the newly formed bone, being the residual biomaterial absent, or minimal). Interestingly, Di Stefano et al.²⁶ still found, 3.5 months after grafting, a significant amount of biomaterial (about 20.5%); instead, in the present study none, or almost none, was observed. Overall, the results of our study and those of Di Stefano et al.²⁶ seem to indicate that the bone substitute under examination is capable of favoring bone formation within a relatively short period; this is consistent with previous clinical and histomorphometric evidence concerning the particulate component this equine bone paste is made of, that is, particulate EDEB.^16,36,37In vitro studies have shown that EDEB’s proregenerating property might be linked to the way osteoclasts interact with it — that is, by adhering and remodeling the equine bone³⁸ in a physiological way (and differently from what they do on ABB³⁹). Most probably, EDEB preserves type I bone collagen unaltered in its native conformation,^15,16,35,38 which, in turn, might allow its modulation of several processes related to bone regeneration.^40–43 These observations are consistent with the histomorphometric ones comparing EDEB and ABB (which is collagen-free):¹² when the two materials were grafted in prospectively recruited and randomly allocated patients who underwent sinus augmentation, biopsies collected at 6 months from sinuses grafted with EDEB systematically contained more newly formed bone and less residual biomaterial than those collected from sinuses grafted with ABB.³⁶ A further confirmation comes from the study by Di Stefano et al. in 46 patients seeking implant-supported rehabilitation and treated with EDEB or ABB.⁴⁴ In that study, no collagen or other proteins were detected in ABB samples by the attenuated total reflection Fourier transform infrared (ATR-FTIR) analysis. On the contrary, both the ATR-FTIR and the SDS-PAGE analyzes showed the presence of collagen in its native conformation in EDEB samples.⁴⁴ Furthermore, histomorphometric examination demonstrated a significantly higher amount of newly formed bone in sites grafted with EDEB compared to those grafted with ABB. Similarly, a fewer amount of residual biomaterial was detected in sites grafted with EDEB.⁴⁴ Another study retrospectively analyzed histomorphometric data of biopsies collected at different times from sinuses grafted with EDEB and concluded that bone formation at 4 months after grafting was not significantly different from those observed at later time points.³⁷ These results are in accordance with the results of the present study, supporting the evidence that native type I bone collagen in EDEB may have favored bone regeneration. The equine bone paste (EDEBEX) used in this pilot investigation also contains vitamin C, which is a requested cofactor of prolyl-hydroxylase and lysyl-hydroxylase, two enzymes that catalyze collagen fibril assembly and are essential during bone formation.⁴⁵ This might further explain the early bone deposition and bone substitute remodeling observed in our study. Interestingly, the histological investigations performed in the present study highlighted a peculiar structure in the inner part of some bone trabeculae, which can be ascribable to hydrogel remnants and, therefore, the original grafting site of the biomaterial. These areas are characterized by a dense concentration of mature osteocytes, possibly suggesting that the grafting material has been fully populated by the cells first, and then acted as the starting point for new bone formation. However, all these observations and speculations should be considered as preliminary. Our histomorphometric data differ from those by Di Stefano et al.²⁶ concerning the amount of residual biomaterial observed; our data concerning patient C, whose biopsy was collected at the latest time point (7 months), showed that bone tissue was still undergoing deposition, while bone remodeling have already reached a balance between anabolism and catabolism in patient B, whose biopsy was collected 1 month earlier. These results indicate that more factors, other than the bone paste, may modulate the kinetic of its remodeling. This should definitively be the subject of further studies. Our observations also confirm that freeze-dried EDEBEX may be used in post-extractive sockets not undergoing any flap preparation, that is — left to heal by second intention. This property, together with its easy-handling, should also be further investigated with appropriate studies.

CONCLUSION

The results of this preliminary, pilot histomorphometric investigation on a novel freeze-dried equine-derived bone paste show that it allows successful socket preservation, promoting the formation of a large amount of bone, therefore reinforcing prior indications that it might be a promising bone graft for ridge preservation surgeries. These preliminary findings should be the subject of further targeted studies.

CLINICAL SIGNIFICANCE

Ridge preservation using bone substitutes as an alternative to autogenous bone is known to be effective, yet available clinical evidence still does not indicate the biomaterial, if any, that should be preferred to carry it out. The equine bone paste used in the present study appears to be a good candidate for further investigation given the bone formation rate it seems to display and its easy-handling property in the clinical setting.

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