Ridge Preservation Using an Innovative Enzyme-deantigenic Equine Bone Paste: A Case Report with 36-month Follow-up

BACKGROUND

After tooth avulsion, the alveolar process undergoes a resorption following a spatial and temporal pattern that has been extensively studied.^1,2 The ridge may resorb to such an extent that implant placement is no more feasible, or implant-supported rehabilitation would be at risk for a poor functional and esthetic outcome.² Ridge contraction may be limited by grafting the post-extractive socket using a biomaterial, acting as a bone substitute, following the ridge preservation³ technique. While an autogenous bone is still being regarded as the gold standard graft, as it contains both living cells and growth factors,⁴ its use exposes the patient to additional intra- and post-surgical risks.⁵ For this reason, many synthetic or natural biomaterials have been proposed as an alternative.⁶ Xenografts (i.e., bone grafts manufactured by processing bone tissue from a non-human mammal) may in principle present a biological advantage over other natural or synthetic grafts because of the similarity in bone tridimensional structure and chemical composition humans shares with the other mammals.⁷ Among xenografts, recent studies have proposed an enzyme-deantigenic equine bone (EDEB) as an alternative to an organic bovine bone (ABB), with the xenograft having the longest history of use in oral surgery and maxillofacial surgery.^8,9 Different from ABB, EDEB is manufactured by subjecting a bovine bone to high temperatures, which eliminates all organic components.¹⁰ EDEB is made non-antigenic by subjecting an equine bone to the action of lytic enzymes and has already been used in different kinds of oral and maxillofacial surgery applications requiring bone grafting.^11–18 It was used effectively also in orthopedic applications.¹⁹ When biomaterials comprising xenografts are used for socket preservation, their handling characteristics may facilitate grafting, helping the surgeon to fill the socket easily and contributing to graft stabilization. Recently, a novel bone paste has been introduced on the market; it consists of a polyethylene glycol/hydroxyl-propyl methyl cellulose-based hydrogel (PEG/HPMC) that acts as a carrier, containing cancellous and cortical EDEB granules and equine-demineralized bone matrix.²⁰ The gel is added with a subsidiary amount of vitamin C acting as a viscomodulator. Bone pastes allow avoiding granule dispersion and loss during surgery, and to assure complete filling and direct contact with the tissue surrounding the defect, possibly facilitating bone repair. It has been shown in an in vitro study on human bone marrow stem cells that this novel equine-derived bone paste stimulates the expression of a known modulator of bone regeneration as the RUNT-related transcription factor 2 (RUNX2), bone bialoprotein (BSP), and osteocalcin.²⁰ The same study also investigated in vivo the effects of this bone paste when grafted in artificially induced femoral defects in rabbits. A histological analysis of the grafted sites at 1 and 2 months showed an interesting reorganization of the regenerating tissue within the lesion boundaries.²⁰ While EDEB has already been used in oral, maxillofacial, and orthopedic surgeries, to the author’s knowledge, no clinical application of this equine bone paste was ever reported in the literature. The aim of the present study is to illustrate the clinical, histological, and histomorphometric outcome relating to a clinical case where this novel bone paste was used successfully to carry out a socket preservation surgery.

CASE DESCRIPTION

The patient was a 45-year-old man with a non-contributory medical history presenting with pain at mastication in his lower right quadrant. Clinical and radiographic examination revealed tooth 4.7, already devitalized and rehabilitated with a ceramic crown, presented with a large carious lesion and was definitively compromised (Fig. 1A). A two-step procedure that included grafting the socket to preserve it from resorption and consequent placement of an implant was developed with the aim of rehabilitating the patient with a single crown. The patient provided informed consent.

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

The tooth was extracted atraumatically (Figs 1B and C) and the socket debrided from any residual of fibrous tissue. Subsequently, the bone paste (Activabone® Mouldable Paste, Bioteck, Arcugnano, Italy, details provided in the following paragraph) was extruded from its syringe directly over the socket (Fig. 1D) and gently pressed with a round instrument to fill the socket (Fig. 1E). No flaps were elevated. After grafting, cross stitches (5-0 non-resorbable suture (Monomyd, Butterfly, Cavenago, Italy)) were applied to stabilize the graft (Fig. 1F). Gingival rims were left open, seeking for second intention healing. Second surgery followed after 3 months. The clinical appearance of soft tissues was quite satisfying (Fig. 2A) as well as the radiographic appearance of the grafted area (Fig. 2B). Antibiotic prophylaxis and post-surgical treatment, anesthetic treatment, and pain management were carried out as in the first intervention. After gaining access to the bone ridge, a bone core (Figs 2C to E) was collected using a trephine and placed in a test tube containing buffered 10% formalin for subsequent histological analysis. The implant site was then prepared following the drilling sequence advised by the manufacturer and a 5 × 10 mm cylindrical implant (Stone, IDI Evolution, Concorezzo, Italy) was positioned and was left non-submerged (Figs 2F and G). A provisional crown was delivered after 3.5 months; definitive crown delivery followed after 1 month. The patient was followed up every month for the following 6 months, and then every 6 months up to 36 months after implant surgery.

RESULTS

The patient healed uneventfully and the final appearance of the rehabilitation was still quite satisfactory (Fig. 3), with no gingival recession evident. Bone levels were maintained between the grafting procedure and implant placement, confirming the socket preservation grafting surgery worked effectively. Bone peri-implant levels were found to be maintained (Fig. 3E) also at the following controls. At the 36-month follow-up, the prosthetic crown was working effectively and the implant met the requirements for success defined by Albrektsson et al.²¹ Qualitative histologic analysis showed a quite extended newly-formed bone area throughout all the sample (Fig. 2H). A small number of residual bone graft particles could be observed because of their affinity for hematoxylin and for having bone lacunae devoid of osteocytes. Residual particles were in close contact with the newly formed bone. No signs of inflammation or cartilage-like tissue could be observed, confirming all the components of the bone paste are fully biocompatible. Lamellar (mature) mineralized bone areas could be observed in most of the sample. Histomorphometric results were NFB = 60.12% and RB = 20.53%.

DISCUSSION

Ridge preservation involves using a bone graft that prevents, at least partially, bone resorption to occur; indeed, Vignoletti et al.²² concluded in 2012 that although some degree of bone modelling and remodeling will occur after tooth extraction, different ridge preservation procedures resulted in significantly less vertical and horizontal contraction of the alveolar bone crest. Yet, although the use of barrier membranes, flap surgical procedure, and full flap closure demonstrated better results, no conclusion could be reached regarding the type of surgical procedure or the most suitable biomaterial. A similar conclusion was reached in 2015 by the Cochrane group,²³ which stated on the basis of their meta-analysis of eight RCTs with a total of 233 extraction sites in 184 participants that there was still no convincing evidence of any clinically significant difference between different grafting materials and barriers used for ridge preservation and, accordingly, that further long term, well-designed RCTs were necessary. The matter is still debated, with investigations trying to determine the most effective graft also resorting to complex statistic meta-analysis models.²⁴ The present case illustrates the successful use of a novel equine-derived bone paste; results were satisfying both considering the short-term clinical outcome and the histomorphometric data showing, at 4 months from the grafting surgery, a significant bone formation inside the socket. No inflammatory reactions could be observed, suggesting a neutral interaction of the bone paste with the newly regenerating bone tissue. This is consistent with previous histological and immunohistochemistry studies on EDEB in bone regeneration in other oral surgery applications.^14,25,26 The significant amount of NFB observed may partially be explained by the behavior of osteoclasts that was observed when they were cultured on EDEB in previous in vitro experiments.²⁷ This behavior may, in turn, be explained by the presence of native collagen in EDEB.^13,14,23,27 Bone collagen is involved in or modulates many processes related to bone regeneration.^28–31 Indeed, a randomized clinical trial on forty patients that compared the use of EDEB and anorganic bovine bone (ABB), not containing any collagen,¹⁰ for maxillary sinus augmentation, showed that grafting EDEB resulted in a greater quantity of newly formed bone (NFB), and a smaller quantity of residual biomaterial (RB) at implant insertion than when ABB was used,²⁵ and a retrospective study investigated bone formation over time following maxillary sinus augmentation using EDEB and showed that new bone formation occurred at an early time (%3C;3 months) after grafting.²⁶ Both results were explained by the claim that EDEB contains type I bone collagen in its native state.

Further, it may be speculated that vitamin C within the hydrogel carrier may sustain collagen deposition throughout the entire lesion volume, vitamin C being a requested co-factor for the activity of prolyl- and lysil-hydroxylases, essential enzymes for collagen fibril assembly.³² An additional advantage of a bone paste with respect to a granular graft is the carrier providing spatial stabilization to granules while at the same time facilitating the contact of the graft with all the surrounding vital bone. The authors appreciated, in particular, the handling properties of the equine bone paste that was firm but, at the same time, easily moldable and could be easily adapted to the socket. Finally, it should be remarked that the positive results experienced in this clinical case were achieved without using any membrane and leaving the socket to heal by second intention.

CONCLUSION

The equine-derived bone paste used in the present case allowed for successful socket preservation, and, taking into consideration its handling properties as well, it might be a promising bone graft to carry out ridge preservation surgeries. Accordingly, its actual effectiveness for this indication should be the subject of further investigations.

CLINICAL SIGNIFICANCE

Alveolar ridge preservation using bone grafts is a well-known approach, yet there is still no agreement about which bone graft might be considered the most suitable for this indication. The novel equine-derived bone paste used in the present study appears a promising option for effective socket preservation and may promote secondary intention healing.

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