Clinical-radiographic and histological evaluation of two hydroxyapatites in human extraction sockets: a pilot study


After tooth extraction the healing process involves bone resorption and soft tissue contraction, events that can compromise the ideal implant placement with functional and aesthetic limitations. Following tooth extraction, a socket preservation technique can limit bone resorption. This study evaluated two different types of hydroxyapatite (HA) grafting materials placed into fresh extraction sockets, 6 months after tooth extraction, histologically, clinically and radiographically. Ten extraction sockets from 10 patients were divided in two groups: 5 sockets received a biomimetic HA and 5 received nanocrystalline HA. After 6 months, before implant placement, samples from the grafted area were harvested and evaluated clinically, radiographically and histologically. The percentages of bone, osteoid areas and residual material in the two groups were not statistically different. All samples showed great variability with extensive bone formation and total material resorption or amounts of osteoid tissue that filled the spaces between the residual material particles. The authors did not find any differences between biomimetic and nanocrystalline HA and assume that, within the limits of this study, both these materials could be applied into fresh extraction sockets to limit bone resorption. A control material and a much larger sample size are needed to confirm these findings.

Trauma, severe periodontitis and endodontic lesions are the most significant reasons for tooth extraction, and endosseous implant placement represents one of the most common solutions for subsequent oral rehabilitation. An atraumatic extraction socket heals uneventfully with newly formed bone tissue 1–2 months after extraction . This healing process often occurs with bone resorption and soft tissue shrinkage, which can compromise the ideal implant placement and the final aesthetics . Controlled clinical studies demonstrated a 4.4 mm average of horizontal bone resorption and 1.2 mm of vertical bone resorption up to 6 months after tooth extraction . A socket preservation technique can limit bone remodelling and prevent tissue collapsing . Several studies have proposed different ridge preservation techniques following tooth extraction, including placement of various graft materials in the socket . Many of these grafting materials used in human extraction sockets, such as demineralized freeze-dried bone allograft (DFDBA), bioactive glass, and deproteinized natural bovine bone, do not reabsorb and remain in situ for a long time after healing. Biopsies from preserved extraction sites before implant placement showed the presence of material particles 6–9 months after their insertion . Preliminary studies showed that nano-sized ceramics could be a promising class of bone substitutes, owing to their improved osseointegration properties . New synthetic products with improved biological performance have been introduced to the market.

A synthetic nanocrystalline hydroxyapatite (HA) paste containing about 65% water and 35% nanostructured apatite particles has been introduced for bone augmentation procedures . The crystalline size of this material is about 18 nm. The advantages of such a nanostructured material are its close contact with surrounding tissues and its quick resorption characteristics. Undisturbed osseous integration and complete resorption of nano-HA paste occurs within 12 weeks .

Another recently introduced product is a new type of nano-HA, called biomimetic HA, in which the crystalline size is about 30–40 nm. Calcium ions are replaced by the magnesium ions that are present in the HA of human bone; this allows the material to mimic human bone and enhances its degradation .

The aim of this study was to perform a histological, clinical and radiographic evaluation of two HA-based materials placed into fresh extraction sockets. The amount and quality of new bone tissue and the resorption rate of the two materials were considered, 6 months after grafting, before implantation.

Materials and methods

Ten consecutive patients (10 females), aged 43–67 years (arithmetic mean plus and minus standard deviation of the mean: 54 ± 8 years; median value: 51 years), and undergoing periodontal treatment, participated in this study. All patients were healthy, with no systemic disease (i.e. diabetes, autoimmune dysfunction, prolonged cortisone therapy or chemotherapy) and did not take any medications that could alter wound healing after extraction. All patients had been non-smokers for at least 12 months. All the patients had at least one tooth to be extracted and replaced with an endosseous implant. Exclusion criteria were: the presence of acute infection around the alveolar bone in the surgical site; coagulation disorders; and inadequate sampling due to the small trephine.

This study was carried out in accordance with the Helsinki Declaration of 1975, as revised in 2000. Before entering the study, all subjects were informed about the nature of the study and they all signed an informed consent form. All the patients received initial periodontal therapy and oral hygiene instructions.

At the time of extraction, the patients were randomly assigned to the test group (T) or the control group (C). Following local anaesthesia (mepivacaine 20 mg/ml with adrenaline 1:100,000), atraumatic extraction was performed and the sockets were debrided. The T sites were filled with granules of a biomimetic hydroxyapatite (T-BHA, SINTLife™, FinCeramica, Faenza, Italy) and the C sites were grafted with nanocrystalline hydroxyapatite putty (C-NHA, Ostim ® , Haraeus Kulzer, Hanau, Germany). Once the material was placed into the socket, it was compressed without excessive force until the bony socket was completely filled. The grafted sites were then covered with a collagen sponge and sutured (Vicryl 4/0, Ethicon, Johnson&Johnson Company) with no attempt to obtain primary closure of the wound.

All patients were instructed to rinse with chlorhexidine 0.2% twice a day and to use analgesics only in case of postoperative pain. No antibiotic was prescribed and sutures were removed after 10 days. Patients were recalled for oral hygiene every 3 months during the monitoring period.

Endosseous implants were placed 6 months after tooth extractions (T-BHA, m ± SD: 6.0 ± 1.6 months; C-NHA, m ± SD: 5.6 ± 1.1 months). After local anaesthesia, a full thickness mucoperiosteal flap was elevated; a bone trephine bur with an external diameter of 2 mm was used instead of the 2 mm diameter twist drill, to take a bone core during preparation of the implant sites. The direction of the trephine drill was guided by an individual acrylic stent. After the harvesting procedure, a landmark was added in order to identify the apical-coronal orientation of the specimens. The surgical site was enlarged and deepened to receive the endosseous implant with a diameter >3 mm and length >8 mm. The biopsies were fixed in 10% formalin solution buffered at pH 7.2 (Bio-Optica, Milan, Italy), dehydrated and embedded in methyl methacrylate.

During the preparation of the surgical site with the different trephines, bone hardness was evaluated using the Misch bone quality scale . Standardized intra-oral radiographs (Kodak Ultra-Speed DF 57, Eastman Kodak Company, NY, USA) were taken immediately after the graft, prior to implant site preparation, and after implant placement. No measurements were performed on the radiographs.

Four to 6 μm serial sections of the resin-embedded fragments were obtained by a rotative microtome (model RM2155 Leica Instruments, Germany), and stained with haematoxylin-eosin (H-E) and Giemsa (Merck, Darmstadt, Germany), as well as with Goldner’s trichromic stain (Bio-Optica, Milan, Italy), in order to identify red-orange osteoid or connective tissue and blue-green mineralized bone.

Two evaluators performed the histopathological evaluation blindly, using a light polarized microscope (Nikon Eclipse E800M, Tokyo, Japan), and considering the biopsies at 100× and 600× original microscope magnification. Low magnification (40×) digital micrographs of the sections were recorded, and a rectangular window to fit the core of the biopsy exactly was identified, in order to perform the histomorphometric analysis. The proportions of mature bone, osteoid, fibrous tissue and residual material were determined ( Fig. 1 ). The measurements were expressed as percentages of the total area of the selected windows. The presence of inflammation and the induction of vascularization were also examined.

Fig. 1
(a) Light micrograph of a C-NHA biopsy at the site of graft insertion (#7504, Giemsa stain, 40×). (b) Light micrograph of a T-BHA biopsy at the site of graft insertion (#7505, Giemsa stain, 40×). Mature bone (Mb) and residual material (M) filling the defect are shown at left the coronal part.

For each patient the histomorphometric data were calculated from five sections on each sample and were expressed as arithmetic mean plus and minus standard deviation of the mean ( m ± SD). The comparison between groups was performed using Student’s t -test; p < 0.05 was considered significant.


All extraction sockets healed uneventfully. No infections were observed during the study period.

On retrieval of the core, clinical measurements were made at each site. The mean clinical hardness of the bone formed from both the T-BHA and the C-NHA treated sites were comparable and ranged between type III and IV bone qualities .

Both biomaterials used in this study showed a similar grade of radiopacity to the surrounding alveolar bone, even though no measurements were performed on the radiographs. Just after the grafting procedure, the biomaterial depicted clearly distinguishable texture for both products and remained more or less evident until the end of the observation period. A limited centripetal volume reduction of the grafted materials was detected in the subsequent radiographic examinations. The volume of the biomaterial seemed to decrease from the bone margins of the defect towards the centre, parallel to the new bone apposition. At 6 months, the residual presence of the grafted materials was still detectable ( Fig. 2 ).

Fig. 2
Radiographs of the sockets. (a) 6-Month healing of the socket of the upper left lateral incisor, grafted with T-BHA. Residual presence of the grafted material is detectable. (b) 6-Month healing of the socket of the upper right second premolar, grafted with C-NHA. Residual presence of the grafted material is detectable.

In three cases, C-NHA patients showed extensive bone formation throughout the defect and total material resorption ( Fig. 3 (a)) . In two cases active bone remodelling and incomplete healing was demonstrated and the material underwent different degrees of partial resorption ( Fig. 3 (b), (c)). Usually, the C-NHA was well integrated in the bone tissue ( Fig. 3 (d)). In three T-BHA patients, active bone remodelling was found and mature bone filled the defect ( Fig. 4 (a) , (c)). In two specimens a discrete amount of osteoid tissue that filled the spaces between the residual material particles was shown ( Fig. 4 (b) and (d)). As in C-NHA, a discrete integration between T-BHA and newly formed bone tissue was present. Rarely, a fibrous-histiocytic reaction was evident. There was no evidence of acute or chronic infection in the samples: polymorphonuclear cells, lymphocytes and plasma cells were always absent.

Fig. 3
Light micrographs of C-NHA biopsies at the site of graft insertion. (a) Mature bone (Mb) and bone marrow-like tissue (Ml) filling the defect, total material resorption (#7585, Giemsa stain, polarized light, 100×). (b) Extensive bone remodelling throughout the defect, osteoid (Os), fibrous tissue (Ft) and partial resorption of the material (M) (#7530, Giemsa stain, 100×). (c) Mature bone (B, blue-green), osteoid tissue (Os, red) and partial resorption of the material (M) (#7530, Goldner’s trichromic stain, 400×). (d) NHA (M) well integrated in the mature bone tissue (Mb) (#7504, Giemsa stain, 200×). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 4
Light micrographs of T-BHA biopsies at the site of graft insertion. (a) Marrow-like tissue (Ml) and mature bone (Mb) filling the defect, scarce residual material (M) (#7505, Giemsa stain, 100×). (b) Active bone remodelling, mature bone (Mb), osteoid seams (Os) and large pieces of material (#7539, Giemsa stain, 100×). (c) Active bone remodelling, marrow-like tissue (Ml) and mature bone (Mb), scarce residual material (M) (#7580, Giemsa stain, 200×). (d) Abundant residual material (M) (#7499, Giemsa stain, 100×). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

The histomorphometric analysis confirmed a large variability amongst all patients in both groups. When Student’s t -test was applied to highlight the specific differences between T-BHA and C-NHA patients, the percentages of bone, osteoid areas, and residual material were not significantly different ( Table 1 ). Detailed results are reported in Fig. 5 .

Table 1
Histomorphometric results. Comparison (Student’s t -test) between patients with nanocrystalline hydroxyapatite putty (C-NHA) grafts and biomimetic hydroxyapatite (T-BHA) grafts. All the values are expressed as m ± SD (median value) and min–max range. p < 0.05 was considered statistically significant.
Histomorphometric parameters C-NHA T-BHA p value
Mature bone (%) 54 ± 22 (60)
49 ± 28 (60)
Osteoid tissue (%) 36 ± 14 (31)
34 ± 24 (26)
Fibrous tissue (%) 3 ± 5 (2)
7 ± 9 (7)
Material residue (%) 8 ± 7 (4)
14 ± 7 (10)

Feb 7, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Clinical-radiographic and histological evaluation of two hydroxyapatites in human extraction sockets: a pilot study
Premium Wordpress Themes by UFO Themes